Celebrating 30 Years

Genetically Engineered Natural Killer Cells in Cancer Therapy: Advances, Challenges, and Future Directions

Expand
  • 1. Department of Hematology; Shanghai Institute of Hematology; National Research Center for Translational Medicine at Shanghai, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; 2. Shanghai Immune Therapy Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China; 3. Collaborative Innovation Center of Hematology, Shanghai Jiao Tong University, Shanghai 200240, China

Received date: 2025-12-23

  Revised date: 2026-03-22

  Accepted date: 2026-04-16

  Online published: 2026-06-11

Abstract

A cure of cancer remains challenging. With the advancements in gene editing techniques, immune cell-based treatments (e.g., natural killer (NK) cell therapy) have emerged as potent therapeutic modalities, aiming to address clinical bottlenecks associated with traditional approaches. This review recapitulates the inherent characteristics of NK cells as a safe allogeneic tool against cancers, as well as available gene-editing systems for enhancing NK-cell activity, improving persistence, and increasing safety. Furthermore, it summarizes the preclinical and clinical practices of genetically engineered NK cell therapies and highlights their potential, challenges, and future perspectives in cancer treatment. Overall, we provide a comprehensive insight into NK cell-based immunotherapies as a promising approach for cancer treatment.

Cite this article

You Yiqi, Dong Han, Xu Jie . Genetically Engineered Natural Killer Cells in Cancer Therapy: Advances, Challenges, and Future Directions[J]. Journal of Shanghai Jiaotong University(Science), 2026 , 31(3) : 584 -603 . DOI: 10.1007/s12204-026-2936-7

References

[1] Wolf N K, Kissiov D U, Raulet D H. Roles of natural killer cells in immunity to cancer, and applications to immunotherapy [J]. Nature Reviews Immunology, 2023, 23(2): 90-105.
[2] Alfarra H, Weir J, Grieve S, et al. Targeting NK cell inhibitory receptors for precision multiple myeloma immunotherapy [J]. Frontiers in Immunology, 2020, 11: 575609.
[3] Pallmer K, Oxenius A. Recognition and regulation of T cells by NK cells [J]. Frontiers in Immunology, 2016, 7: 251.
[4] Coënon L, Geindreau M, Ghiringhelli F, et al. Natural Killer cells at the frontline in the fight against cancer [J]. Cell Death & Disease, 2024, 15: 614.
[5] Ma S B, Yu J H, Caligiuri M A. Natural killer cell–based immunotherapy for cancer [J]. The Journal of Immunology, 2025, 214(7): 1444-1456.
[6] Zhang A Q, Yang X C, Zhang Y C, et al. Unlocking the potential of CAR-NK cell therapy: Overcoming barriers and challenges in the treatment of myeloid malignancies [J]. Molecular Cancer Therapeutics, 2025, 24(4): 536-549.
[7] Fujisaki H, Kakuda H, Shimasaki N, et al. Expansion of highly cytotoxic human natural killer cells for cancer cell therapy [J]. Cancer Research, 2009, 69(9): 4010-4017.
[8] Grzywacz B, Moench L, McKenna D, et al. Natural killer cell homing and persistence in the bone marrow after adoptive immunotherapy correlates with better leukemia control [J]. Journal of Immunotherapy, 2019, 42(2): 65-72.
[9] Björklund A T, Carlsten M, Sohlberg E, et al. Complete remission with reduction of high-risk clones following haploidentical NK-cell therapy against MDS and AML [J]. Clinical Cancer Research, 2018, 24(8): 1834-1844.
[10] Denman C J, Senyukov V V, Somanchi S S, et al. Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells [J]. PLoS One, 2012, 7(1): e30264.
[11] Zhang D, Zheng Y S, Lin Z G, et al. Equipping natural killer cells with specific targeting and checkpoint blocking aptamers for enhanced adoptive immunotherapy in solid tumors [J]. Angewandte Chemie International Edition, 2020, 59(29): 12022-12028.
[12] Murray S, Lundqvist A. Targeting the tumor microenvironment to improve natural killer cell-based immunotherapies: On being in the right place at the right time, with resilience [J]. Human Vaccines & Immunotherapeutics, 2016, 12(3): 607-611.
[13] Lu C G, Guo C J, Chen H, et al. A novel chimeric PD1-NKG2D-41BB receptor enhances antitumor activity of NK92 cells against human lung cancer H1299 cells by triggering pyroptosis [J]. Molecular Immunology, 2020, 122: 200-206.
[14] Li T X, Yang Y Y, Qi H Z, et al. CRISPR/Cas9 therapeutics: Progress and prospects [J]. Signal Transduction and Targeted Therapy, 2023, 8: 36.
[15] Li T Y, Li S Q, Kang Y, et al. Harnessing the evolving CRISPR/Cas9 for precision oncology [J]. Journal of Translational Medicine, 2024, 22(1): 749.
[16] Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity [J]. Science, 2012, 337(6096): 816-821.
[17] Zhang X H, Tee L Y, Wang X G, et al. Off-target effects in CRISPR/Cas9-mediated genome engineering [J]. Molecular Therapy - Nucleic Acids, 2015, 4: e264.
[18] Bhardwaj A, Nain V. TALENs: An indispensable tool in the era of CRISPR: A mini review [J]. Journal of Genetic Engineering and Biotechnology, 2021, 19(1): 125.
[19] Kampmann M. CRISPR-based functional genomics for neurological disease [J]. Nature Reviews Neurology, 2020, 16(9): 465-480.
[20] Kanafi M M, Tavallaei M. Overview of advances in CRISPR/deadCas9 technology and its applications in human diseases [J]. Gene, 2022, 830: 146518.
[21] Enright A L, Heelan W J, Ward R D, et al. CRISPRi functional genomics in bacteria and its application to medical and industrial research [J]. Microbiology and Molecular Biology Reviews, 2024, 88(2): e00170-22.
[22] Villegas Kcam M C, Tsong A J, Chappell J. Uncovering the distinct properties of a bacterial type I-E CRISPR activation system [J]. ACS Synthetic Biology, 2022, 11(2): 1000-1003.
[23] Cui X L, Zhang C, Xu Z F, et al. Dual CRISPR interference and activation for targeted reactivation of X-linked endogenous FOXP3 in human breast cancer cells [J]. Molecular Cancer, 2022, 21(1): 38.
[24] Gaudelli N M, Komor A C, Rees H A, et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage [J]. Nature, 2017, 551(7681): 464-471.
[25] Anzalone A V, Randolph P B, Davis J R, et al. Search-and-replace genome editing without double-strand breaks or donor DNA [J]. Nature, 2019, 576(7785): 149-157.
[26] Chen P J, Liu D R. Prime editing for precise and highly versatile genome manipulation [J]. Nature Reviews Genetics, 2023, 24(3): 161-177.
[27] Gehrke J M, Cervantes O, Clement M K, et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities [J]. Nature Biotechnology, 2018, 36(10): 977-982.
[28] Kwon J, Kim M, Bae S, et al. TAPE-seq is a cell-based method for predicting genome-wide off-target effects of prime editor [J]. Nature Communications, 2022, 13: 7975.
[29] Liang S Q, Liu P P, Ponnienselvan K, et al. Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag [J]. Nature Methods, 2023, 20(6): 898-907.
[30] Yu Z X, Lu Z K, Li J J, et al. PEAC-seq adopts Prime Editor to detect CRISPR off-target and DNA translocation [J]. Nature Communications, 2022, 13: 7545.
[31] González Castro N, Bjelic J, Malhotra G, et al. Comparison of the feasibility, efficiency, and safety of genome editing technologies [J]. International Journal of Molecular Sciences, 2021, 22(19): 10355.
[32] Chou S T, Leng Q X, Mixson A J. Zinc finger nucleases: Tailor-made for gene therapy [J]. Drugs of the Future, 2012, 37(3): 183-196.
[33] Gupta D, Bhattacharjee O, Mandal D, et al. CRISPR-Cas9 system: A new-fangled dawn in gene editing [J]. Life Sciences, 2019, 232: 116636.
[34] Tang S Y, Zha S J, Du Z C, et al. Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells [J]. Stem Cell Research & Therapy, 2021, 12(1): 580.
[35] Richter A, Streubel J, Boch J. TAL effector DNA-binding principles and specificity [J]. Methods in Molecular Biology, 2016, 1338: 9-25.
[36] Deng D, Yan C Y, Pan X J, et al. Structural basis for sequence-specific recognition of DNA by TAL effectors [J]. Science, 2012, 335(6069): 720-723.
[37] Li H Y, Yang Y, Hong W Q, et al. Applications of genome editing technology in the targeted therapy of human diseases: Mechanisms, advances and prospects [J]. Signal Transduction and Targeted Therapy, 2020, 5: 1.
[38] Chen A P, Gao P, Ashok P, et al. TALEN®-based gene edited iPSC-derived NK (iNK) cells demonstrate enhanced antitumor activity [J]. Journal for ImmunoTherapy of Cancer, 2022, 10: 323.
[39] Vargas J E, Chicaybam L, Stein R T, et al. Retroviral vectors and transposons for stable gene therapy: Advances, current challenges and perspectives [J]. Journal of Translational Medicine, 2016, 14(1): 288.
[40] Geurts A M, Yang Y, Clark K J, et al. Gene transfer into genomes of human cells by the sleeping beauty transposon system [J]. Molecular Therapy, 2003, 8(1): 108-117.
[41] Turchiano G, Latella M C, Gogol-Döring A, et al. Genomic analysis of Sleeping Beauty transposon integration in human somatic cells [J]. PLoS One, 2014, 9(11): e112712.
[42] Bire S, Casteret S, Arnaoty A, et al. Transposase concentration controls transposition activity: Myth or reality? [J]. Gene, 2013, 530(2): 165-171.
[43] Izsvák Z, Ivics Z. Sleeping beauty transposition: Biology and applications for molecular therapy [J]. Molecular Therapy, 2004, 9(2): 147-156.
[44] Ding S, Wu X H, Li G, et al. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice [J]. Cell, 2005, 122(3): 473-483.
[45] Li M A, Turner D J, Ning Z M, et al. Mobilization of giant piggyBac transposons in the mouse genome [J]. Nucleic Acids Research, 2011, 39(22): e148.
[46] Cadiñanos J, Bradley A. Generation of an inducible and optimized piggyBac transposon system [J]. Nucleic Acids Research, 2007, 35(12): e87.
[47] Sun Y, Liu G, Huang Y. Applications of piggyBac transposons for genome manipulation in stem cells [J]. Stem Cells International, 2021, 2021(1): 3829286.
[48] Li M D, Bronson D L, Lemke T D, et al. Phylogenetic analyses of 55 retroelements on the basis of the nucleotide and product amino acid sequences of the pol gene [J]. Molecular Biology and Evolution, 1995, 12(4): 657-670.
[49] Fang M G, Allen A, Luo C, et al. Unlocking the potential of iPSC-derived immune cells: Engineering iNK and iT cells for cutting-edge immunotherapy [J]. Frontiers in Immunology, 2024, 15: 1457629.
[50] Goldenson B H, Hor P, Kaufman D S. iPSC-derived natural killer cell therapies - expansion and targeting [J]. Frontiers in Immunology, 2022, 13: 841107.
[51] Knorr D A, Ni Z Y, Hermanson D, et al. Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy [J]. Stem Cells Translational Medicine, 2013, 2(4): 274-283.
[52] Woll P S, Martin C H, Miller J S, et al. Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity [J]. Journal of Immunology, 2005, 175(8): 5095-5103.
[53] Wang X B, Zhang Y, Jin Y, et al. An iPSC-derived CD19/BCMA CAR-NK therapy in a patient with systemic sclerosis [J]. Cell, 2025, 188(16): 4225-4238.e12.
[54] Wu X Y, Matosevic S. Gene-edited and CAR-NK cells: Opportunities and challenges with engineering of NK cells for immunotherapy [J]. Molecular Therapy - Oncolytics, 2022, 27: 224-238.
[55] Müller S, Bexte T, Gebel V, et al. High cytotoxic efficiency of lentivirally and alpharetrovirally engineered CD19-specific chimeric antigen receptor natural killer cells against acute lymphoblastic leukemia [J]. Frontiers in Immunology, 2020, 10: 3123.
[56] Leivas A, Valeri A, Córdoba L, et al. NKG2D-CAR-transduced natural killer cells efficiently target multiple myeloma [J]. Blood Cancer Journal, 2021, 11: 146.
[57] Liu E L, Marin D, Banerjee P, et al. Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors [J]. New England Journal of Medicine, 2020, 382(6): 545-553.
[58] Marin D, Li Y, Basar R, et al. Safety, efficacy and determinants of response of allogeneic CD19-specific CAR-NK cells in CD19+ B cell tumors: A phase 1/2 trial [J]. Nature Medicine, 2024, 30(3): 772-784.
[59] Bexte T, Albinger N, Al Ajami A, et al. CRISPR/Cas9 editing of NKG2A improves the efficacy of primary CD33-directed chimeric antigen receptor natural killer cells [J]. Nature Communications, 2024, 15: 8439.
[60] Albinger N, Pfeifer R, Nitsche M, et al. Primary CD33-targeting CAR-NK cells for the treatment of acute myeloid leukemia [J]. Blood Cancer Journal, 2022, 12: 61.
[61] Bexte T, Alzubi J, Reindl L M, et al. CRISPR-Cas9 based gene editing of the immune checkpoint NKG2A enhances NK cell mediated cytotoxicity against multiple myeloma [J]. OncoImmunology, 2022, 11(1): 2081415.
[62] Lin Y Q, Xiao Z Y, Hu F X, et al. Engineered CRO-CD7 CAR-NK cells derived from pluripotent stem cells avoid fratricide and efficiently suppress human T-cell malignancies [J]. Journal of Hematology & Oncology, 2025, 18(1): 57.
[63] Gomes-Silva D, Srinivasan M, Sharma S, et al. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies [J]. Blood, 2017, 130(3): 285-296.
[64] Peng L, Renauer P A, Sferruzza G, et al. In vivo AAV–SB-CRISPR screens of tumor-infiltrating primary NK cells identify genetic checkpoints of CAR-NK therapy [J]. Nature Biotechnology, 2025, 43(5): 752-761.
[65] Vivier E, Raulet D H, Moretta A, et al. Innate or adaptive immunity? The example of natural killer cells [J]. Science, 2011, 331(6013): 44-49.
[66] Floros T, Tarhini A A. Anticancer cytokines: Biology and clinical effects of interferon-α2, interleukin (IL)-2, IL-15, IL-21, and IL-12 [J]. Seminars in Oncology, 2015, 42(4): 539-548.
[67] Nagashima S, Mailliard R, Kashii Y, et al. Stable transduction of the interleukin-2 gene into human natural killer cell lines and their phenotypic and functional characterization in vitro and in vivo [J]. Blood, 1998, 91(10): 3850-3861.
[68] Ranson T, Vosshenrich C A J, Corcuff E, et al. IL-15 is an essential mediator of peripheral NK-cell homeostasis [J]. Blood, 2003, 101(12): 4887-4893.
[69] Wrangle J M, Velcheti V, Patel M R, et al. ALT-803, an IL-15 superagonist, in combination with nivolumab in patients with metastatic non-small cell lung cancer: A non-randomised, open-label, phase 1b trial [J]. The Lancet Oncology, 2018, 19(5): 694-704.
[70] Liu E, Tong Y, Dotti G, et al. Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity [J]. Leukemia, 2018, 32(2): 520-531.
[71] Xu X D, Cao P Y, Wang M, et al. Signaling intact membrane-bound IL-15 enables potent anti-tumor activity and safety of CAR-NK cells [J]. Frontiers in Immunology, 2025, 16: 1658580.
[72] Du Z C, Ng Y Y, Zha S J, et al. piggyBac system to co-express NKG2D CAR and IL-15 to augment the in vivo persistence and anti-AML activity of human peripheral blood NK cells [J]. Molecular Therapy - Methods & Clinical Development, 2021, 23: 582-596.
[73] Feng D D, Sun L, Hu D X, et al. Expression of membrane-bound Interleukin-15 sustains the growth and survival of CAR-NK cells [J]. International Immunopharmacology, 2025, 166: 115577.
[74] Miller J S, Morishima C, McNeel D G, et al. A first-in-human phase I study of subcutaneous outpatient recombinant human IL15 (rhIL15) in adults with advanced solid tumors [J]. Clinical Cancer Research, 2018, 24(7): 1525-1535.
[75] Jamali A, Hadjati J, Madjd Z, et al. Highly efficient generation of transgenically augmented CAR NK cells overexpressing CXCR4 [J]. Frontiers in Immunology, 2020, 11: 2028.
[76] Schomer N T, Jiang Z K, Lloyd M I, et al. CCR7 expression in CD19 chimeric antigen receptor-engineered natural killer cells improves migration toward CCL19-expressing lymphoma cells and increases tumor control in mice with human lymphoma [J]. Cytotherapy, 2022, 24(8): 827-834.
[77] Feigl F F, Stahringer A, Peindl M, et al. Efficient redirection of NK cells by genetic modification with chemokine receptors CCR4 and CCR2B [J]. International Journal of Molecular Sciences, 2023, 24(4): 3129.
[78] Lee N, Llano M, Carretero M, et al. HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A [J]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(9): 5199-5204.
[79] André P, Denis C, Soulas C, et al. Anti-NKG2A MAb is a checkpoint inhibitor that promotes anti-tumor immunity by unleashing both T and NK cells [J]. Cell, 2018, 175(7): 1731-1743.e13.
[80] McWilliams E M, Mele J M, Cheney C, et al. Therapeutic CD94/NKG2A blockade improves natural killer cell dysfunction in chronic lymphocytic leukemia [J]. OncoImmunology, 2016, 5(10): e1226720.
[81] Cózar B, Greppi M, Carpentier S, et al. Tumor-infiltrating natural killer cells [J]. Cancer Discovery, 2021, 11(1): 34-44.
[82] Kamiya T, Seow S V, Wong D, et al. Blocking expression of inhibitory receptor NKG2A overcomes tumor resistance to NK cells [J]. Journal of Clinical Investigation, 2019, 129(5): 2094-2106.
[83] da Silva I P, Gallois A, Jimenez-Baranda S, et al. Reversal of NK-cell exhaustion in advanced melanoma by tim-3 blockade [J]. Cancer Immunology Research, 2014, 2(5): 410-422.
[84] Xu L Y, Huang Y Y, Tan L L, et al. Increased Tim-3 expression in peripheral NK cells predicts a poorer prognosis and Tim-3 blockade improves NK cell-mediated cytotoxicity in human lung adenocarcinoma [J]. International Immunopharmacology, 2015, 29(2): 635-641.
[85] Wang Z X, Zhu J L, Gu H D, et al. The clinical significance of abnormal tim-3 expression on NK cells from patients with gastric cancer [J]. Immunological Investigations, 2015, 44(6): 578-589.
[86] Sakuishi K, Apetoh L, Sullivan J M, et al. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity [J]. Journal of Experimental Medicine, 2010, 207(10): 2187-2194.
[87] Pu F F, Chen F X, Zhang Z C, et al. TIM-3 expression and its association with overall survival in primary osteosarcoma [J]. Oncology Letters, 2019, 18(5): 5294-5300. 
[88] Gallois A, Silva I, Osman I, et al. Reversal of natural killer cell exhaustion by TIM-3 blockade [J]. OncoImmunology, 2014, 3(12): e946365.
[89] Hou H Y, Liu W Y, Wu S J, et al. Tim-3 negatively mediates natural killer cell function in LPS-induced endotoxic shock [J]. PLoS One, 2014, 9(10): e110585.
[90] Zhang Q, Bi J C, Zheng X D, et al. Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity [J]. Nature Immunology, 2018, 19(7): 723-732.
[91] Masson D, Jarry A, Baury B, et al. Overexpression of the CD155 gene in human colorectal carcinoma [J]. Gut, 2001, 49(2): 236-240.
[92] Li M, Xia P Y, Du Y, et al. T-cell immunoglobulin and ITIM domain (TIGIT) receptor/poliovirus receptor (PVR) ligand engagement suppresses interferon-γ production of natural killer cells via β-arrestin 2-mediated negative signaling [J]. Journal of Biological Chemistry, 2014, 289(25): 17647-17657.
[93] Carlsten M, Norell H, Bryceson Y T, et al. Primary human tumor cells expressing CD155 impair tumor targeting by down-regulating DNAM-1 on NK cells [J]. The Journal of Immunology, 2009, 183(8): 4921-4930.
[94] Sarhan D, Cichocki F, Zhang B, et al. Adaptive NK cells with low TIGIT expression are inherently resistant to myeloid-derived suppressor cells [J]. Cancer Research, 2016, 76(19): 5696-5706.
[95] Dong H D, Strome S E, Salomao D R, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune evasion [J]. Nature Medicine, 2002, 8(8): 793-800.
[96] Pesce S, Greppi M, Grossi F, et al. PD/1-PD-ls checkpoint: Insight on the potential role of NK cells [J]. Frontiers in Immunology, 2019, 10: 1242.
[97] Quatrini L, Vacca P, Tumino N, et al. Glucocorticoids and the cytokines IL-12, IL-15, and IL-18 present in the tumor microenvironment induce PD-1 expression on human natural killer cells [J]. Journal of Allergy and Clinical Immunology, 2021, 147(1): 349-360.
[98] Dong Y N, Sun Q, Zhang X W. PD-1 and its ligands are important immune checkpoints in cancer [J]. Oncotarget, 2017, 8(2): 2171-2186.
[99] Benson D M Jr, Bakan C E, Mishra A, et al. The PD-1/PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: A therapeutic target for CT-011, a novel monoclonal anti–PD-1 antibody [J]. Blood, 2010, 116(13): 2286-2294.
[100] Vari F, Arpon D, Keane C, et al. Immune evasion via PD-1/PD-L1 on NK cells and monocyte/macrophages is more prominent in Hodgkin lymphoma than DLBCL [J]. Blood, 2018, 131(16): 1809-1819.
[101] Poznanski S M, Singh K, Ritchie T M, et al. Metabolic flexibility determines human NK cell functional fate in the tumor microenvironment [J]. Cell Metabolism, 2021, 33(6): 1205-1220.e5.
[102] Husain Z, Huang Y N, Seth P, et al. Tumor-derived lactate modifies antitumor immune response: Effect on myeloid-derived suppressor cells and NK cells [J]. The Journal of Immunology, 2013, 191(3): 1486-1495.
[103] Zheng X H, Qian Y B, Fu B Q, et al. Mitochondrial fragmentation limits NK cell-based tumor immunosurveillance [J]. Nature Immunology, 2019, 20(12): 1656-1667.
[104] Allard B, Longhi M S, Robson S C, et al. The ectonucleotidases CD39 and CD73: Novel checkpoint inhibitor targets [J]. Immunological Reviews, 2017, 276(1): 121-144.
[105] Chambers A M, Wang J, Lupo K B, et al. Adenosinergic signaling alters natural killer cell functional responses [J]. Frontiers in Immunology, 2018, 9: 2533.
[106] Pataskar A, Champagne J, Nagel R, et al. Tryptophan depletion results in tryptophan-to-phenylalanine substitutants [J]. Nature, 2022, 603(7902): 721-727.
[107] Della Chiesa M, Carlomagno S, Frumento G, et al. The tryptophan catabolite l-kynurenine inhibits the surface expression of NKp46- and NKG2D-activating receptors and regulates NK-cell function [J]. Blood, 2006, 108(13): 4118-4125.
[108] Michelet X, Dyck L, Hogan A, et al. Metabolic reprogramming of natural killer cells in obesity limits antitumor responses [J]. Nature Immunology, 2018, 19(12): 1330-1340.
[109] O’Brien K L, Finlay D K. Immunometabolism and natural killer cell responses [J]. Nature Reviews Immunology, 2019, 19(5): 282-290.
[110] Ciurea S O, Schafer J R, Bassett R, et al. Phase 1 clinical trial using mbIL21 ex vivo–expanded donor-derived NK cells after haploidentical transplantation [J]. Blood, 2017, 130(16): 1857-1868.
[111] Viel S, Marçais A, Guimaraes F S, et al. TGF-β inhibits the activation and functions of NK cells by repressing the mTOR pathway [J]. Science Signaling, 2016, 9(415): ra19.
[112] Zaiatz-Bittencourt V, Finlay D K, Gardiner C M. Canonical TGF-β signaling pathway represses human NK cell metabolism [J]. The Journal of Immunology, 2018, 200(12): 3934-3941.
[113] Yvon E S, Burga R, Powell A, et al. Cord blood natural killer cells expressing a dominant negative TGF-β receptor: Implications for adoptive immunotherapy for glioblastoma [J]. Cytotherapy, 2017, 19(3): 408-418.
[114] Shaim H, Shanley M, Basar R, et al. Targeting the αv integrin/TGF-β axis improves natural killer cell function against glioblastoma stem cells [J]. Journal of Clinical Investigation, 2021, 131(14): e142116.
[115] Kim T D, Lee S U, Yun S, et al. Human microRNA-27a* targets Prf1 and GzmB expression to regulate NK-cell cytotoxicity [J]. Blood, 2011, 118(20): 5476-5486.
[116] Delconte R B, Kolesnik T B, Dagley L F, et al. CIS is a potent checkpoint in NK cell–mediated tumor immunity [J]. Nature Immunology, 2016, 17(7): 816-824.
[117] Bernard P L, Delconte R, Pastor S, et al. Targeting CISH enhances natural cytotoxicity receptor signaling and reduces NK cell exhaustion to improve solid tumor immunity [J]. Journal for ImmunoTherapy of Cancer, 2022, 10(5): e004244.
[118] Zhu H, Blum R H, Bernareggi D, et al. Metabolic reprograming via deletion of CISH in human iPSC-derived NK cells promotes in vivo persistence and enhances anti-tumor activity [J]. Cell Stem Cell, 2020, 27(2): 224-237.e6.
[119] Donnelly R P, Loftus R M, Keating S E, et al. mTORC1-dependent metabolic reprogramming is a prerequisite for NK cell effector function [J]. The Journal of Immunology, 2014, 193(9): 4477-4484.
[120] Rafei H, Basar R, Acharya S, et al. CREM is a regulatory checkpoint of CAR and IL-15 signalling in NK cells [J]. Nature, 2025, 643(8073): 1076-1086.
[121] Wang M J, Krueger J B, Gilkey A K, et al. Precision enhancement of CAR-NK cells through non-viral engineering and highly multiplexed base editing [J]. Journal for ImmunoTherapy of Cancer, 2025, 13(5): e009560.
[122] Christodoulou I, Ho W J, Marple A, et al. Engineering CAR-NK cells to secrete IL-15 sustains their anti-AML functionality but is associated with systemic toxicities [J]. Journal for ImmunoTherapy of Cancer, 2021, 9(12): e003894.
[123] Di Stasi A, Tey S K, Dotti G, et al. Inducible apoptosis as a safety switch for adoptive cell therapy [J]. New England Journal of Medicine, 2011, 365(18): 1673-1683.
[124] Boztug K, Schmidt M, Schwarzer A, et al. Stem-cell gene therapy for the Wiskott-Aldrich syndrome [J]. The New England Journal of Medicine, 2010, 363(20): 1918-1927.
[125] Bonini C, Ferrari G, Verzeletti S, et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia [J]. Science, 1997, 276(5319): 1719-1724.
[126] Ciceri F, Bonini C, Stanghellini M T L, et al. Infusion of suicide-gene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the TK007 trial): A non-randomised phase I–II study [J]. The Lancet Oncology, 2009, 10(5): 489-500.
[127] Sheikh S, Ernst D, Keating A. Prodrugs and prodrug-activated systems in gene therapy [J]. Molecular Therapy, 2021, 29(5): 1716-1728.
[128] Cloughesy T F, Petrecca K, Walbert T, et al. Effect of vocimagene amiretrorepvec in combination with flucytosine vs standard of care on survival following tumor resection in patients with recurrent high-grade glioma: A randomized clinical trial [J]. JAMA Oncology, 2020, 6(12): 1939-1946.
[129] Fan L F, Freeman K W, Khan T, et al. Improved artificial death switches based on caspases and FADD [J]. Human Gene Therapy, 1999, 10(14): 2273-2285.
[130] Li P, Zhou L B, Zhao T, et al. Caspase-9: Structure, mechanisms and clinical application [J]. Oncotarget, 2017, 8(14): 23996-24008.
[131] Lei W, Liu H, Deng W H, et al. Safety and feasibility of 4-1BB co-stimulated CD19-specific CAR-NK cell therapy in refractory/relapsed large B cell lymphoma: A phase 1 trial [J]. Nature Cancer, 2025, 6(5): 786-800.
[132] Carfagnini C, Singh R, Bechara S B, et al. The efficacy and safety of CD-19 directed CAR-NK therapy in adults with B-cell malignancies: A meta-analysis [J]. Blood, 2024, 144: 7180.
[133] Xu J, Wang B Y, Yu S H, et al. Long-term remission and survival in patients with relapsed or refractory multiple myeloma after treatment with LCAR-B38M CAR T cells: 5-year follow-up of the LEGEND-2 trial [J]. Journal of Hematology & Oncology, 2024, 17(1): 23.
[134] Jagannath S, Martin T G, Lin Y, et al. Long-term (≥5-year) remission and survival after treatment with ciltacabtagene autoleucel in CARTITUDE-1 patients with relapsed/refractory multiple myeloma [J]. Journal of Clinical Oncology, 2025, 43(25): 2766-2771.
[135] Park E, Mun H J, Seo E, et al. CAR NK92 cells targeting BCMA can effectively kill multiple myeloma cells both in vitro and in vivo [J]. Biomedicines, 2024, 12(1): 248.
[136] Motais B, Charvátová S, Walek Z, et al. NK92 expressing anti-BCMA CAR and secreted TRAIL for the treatment of multiple myeloma: Preliminary in vitro assessment [J]. Cells, 2023, 12(23): 2748.
[137] Bras A E, de Haas V, van Stigt A, et al. CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping [J]. Cytometry Part B: Clinical Cytometry, 2019, 96(2): 134-142.
[138] Testa U, Pelosi E, Frankel A. CD 123 is a membrane biomarker and a therapeutic target in hematologic malignancies [J]. Biomarker Research, 2014, 2(1): 4.
[139] Caruso S, De Angelis B, Del Bufalo F, et al. Safe and effective off-the-shelf immunotherapy based on CAR.CD123-NK cells for the treatment of acute myeloid leukaemia [J]. Journal of Hematology & Oncology, 2022, 15(1): 163.
[140] Murakami T, Nakazawa T, Natsume A, et al. Novel human NK cell line carrying CAR targeting EGFRvIII induces antitumor effects in glioblastoma cells [J]. Anticancer Research, 2018, 38(9): 5049-5056.
[141] Zhang C C, Burger M C, Jennewein L, et al. ErbB2/HER2-specific NK cells for targeted therapy of glioblastoma [J]. JNCI: Journal of the National Cancer Institute, 2016, 108(5): djv375.
[142] Liu Y, Zhou Y H, Huang K H, et al. Targeting epidermal growth factor-overexpressing triple-negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor [J]. Cell Proliferation, 2020, 53(8): e12858.
[143] Hu Z W. Tissue factor as a new target for CAR-NK cell immunotherapy of triple-negative breast cancer [J]. Scientific Reports, 2020, 10: 2815.
[144] Schönfeld K, Sahm C, Zhang C C, et al. Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor [J]. Molecular Therapy, 2015, 23(2): 330-338.
[145] Wu M D, He J, Geng J X, et al. Robo1 CAR-NK92 and radiotherapy exert synergistic efficacy in solid tumors [J]. Journal of Translational Medicine, 2025, 23(1): 720.
[146] Yu M, Luo H, Fan M L, et al. Development of GPC3-specific chimeric antigen receptor-engineered natural killer cells for the treatment of hepatocellular carcinoma [J]. Molecular Therapy, 2018, 26(2): 366-378.
[147] Quintarelli C, Sivori S, Caruso S, et al. Efficacy of third-party chimeric antigen receptor modified peripheral blood natural killer cells for adoptive cell therapy of B-cell precursor acute lymphoblastic leukemia [J]. Leukemia, 2020, 34(4): 1102-1115.
[148] Ng Y Y, Du Z C, Zhang X, et al. CXCR4 and anti-BCMA CAR co-modified natural killer cells suppress multiple myeloma progression in a xenograft mouse model [J]. Cancer Gene Therapy, 2022, 29(5): 475-483.
[149] Ren Q, Zu Y L, Su H C, et al. Single VHH-directed BCMA CAR-NK cells for multiple myeloma [J]. Experimental Hematology & Oncology, 2023, 12(1): 98.
[150] Chen K H, Wada M, Firor A E, et al. Novel anti-CD3 chimeric antigen receptor targeting of aggressive T cell malignancies [J]. Oncotarget, 2016, 7(35): 56219-56232.
[151] Zu Y L, Ren Q, Zhang J S, et al. Targeting CD5 chimeric antigen receptor-engineered natural killer cells against T-cell malignancies [J]. Experimental Hematology & Oncology, 2024, 13(1): 104.
[152] You F T, Wang Y Y, Jiang L C, et al. A novel CD7 chimeric antigen receptor-modified NK-92MI cell line targeting T-cell acute lymphoblastic leukemia [J]. American Journal of Cancer Research, 2019, 9(1): 64-78.
[153] Teng K Y, Mansour A G, Zhu Z, et al. Off-the-shelf prostate stem cell antigen–directed chimeric antigen receptor natural killer cell therapy to treat pancreatic cancer [J]. Gastroenterology, 2022, 162(4): 1319-1333.
[154] Han J F, Chu J H, Keung Chan W, et al. CAR-engineered NK cells targeting wild-type EGFR and EGFRvIII enhance killing of glioblastoma and patient-derived glioblastoma stem cells [J]. Scientific Reports, 2015, 5: 11483.
[155] Müller N, Michen S, Tietze S, et al. Engineering NK cells modified with an EGFRvIII-specific chimeric antigen receptor to overexpress CXCR4 improves immunotherapy of CXCL12/SDF-1α-secreting glioblastoma [J]. Journal of Immunotherapy, 2015, 38(5): 197-210.
[156] Klapdor R, Wang S, Morgan M, et al. Characterization of a novel third-generation anti-CD24-CAR against ovarian cancer [J]. International Journal of Molecular Sciences, 2019, 20(3): 660.
[157] Klapdor R, Wang S, Morgan M A, et al. NK cell-mediated eradication of ovarian cancer cells with a novel chimeric antigen receptor directed against CD44 [J]. Biomedicines, 2021, 9(10): 1339.
[158] Klapdor R, Wang S, Hacker U, et al. Improved killing of ovarian cancer stem cells by combining a novel chimeric antigen receptor–based immunotherapy and chemotherapy [J]. Human Gene Therapy, 2017, 28(10): 886-896.
[159] Dickinson M, Hamad N, Bryant C, et al. s261: First in human data of nkx019, an allogeneic car nk for the treatment of relapsed/refractory (r/r) b-cell malignancies [J]. HemaSphere, 2023, 7(S3): e37234fb.
[160] Bachanova V, Ghobadi A, Patel K, et al. Safety and efficacy of FT596, a first-in-class, multi-antigen targeted, off-the-shelf, iPSC-derived CD19 CAR NK cell therapy in relapsed/refractory B-cell lymphoma [J]. Blood, 2021, 138: 823.
[161] Huang R H, Wen Q, Wang X Q, et al. Off-the-shelf CD33 CAR-NK cell therapy for relapse/refractory AML: First-in-human, phase I trial [J]. Blood, 2022, 140(Supplement 1): 7450-7451.
[162] Tang X, Yang L, Li Z, et al. First-in-man clinical trial of CAR NK-92 cells: Safety test of CD33-CAR NK-92 cells in patients with relapsed and refractory acute myeloid leukemia [J]. American Journal of Cancer Research, 2018, 8(6): 1083-1089.
[163] Li B, Zhu X D, Ge J Y, et al. Intraperitoneal infusion of NKG2D CAR-NK cells induces endogenous CD8+ T cell activation in patients with advanced colorectal cancer [J]. Molecular Therapy, 2025, 33(9): 4509-4528.
[164] Sauter C S, Borthakur G, Mountjoy L, et al. A phase 1 study of NKX101, a chimeric antigen receptor natural killer (CAR-NK) cell therapy, with fludarabine and cytarabine in patients with acute myeloid leukemia [J]. Blood, 2023, 142: 2097.
[165] Xiao L, Cen D Z, Gan H N, et al. Adoptive transfer of NKG2D CAR mRNA-engineered natural killer cells in colorectal cancer patients [J]. Molecular Therapy, 2019, 27(6): 1114-1125.
[166] Seery T E, Nangia C S, Reid P D, et al. Overall survival in patients with metastatic or locally advanced pancreatic cancer following chemoradiation with novel combination of aldoxorubicin, N-803 IL-15 superagonist, and PDL1- NK cell therapy [J]. Journal of Clinical Oncology, 2023, 41(4_suppl): 720.
[167] Seery T E, Kistler M, Lee J H, et al. Quilt-3.064: An open-label phase I study of PD-L1 t-haNK in subjects with locally advanced or metastatic solid cancers [J]. Journal of Clinical Oncology, 2020, 38(15_suppl): TPS3152.
[168] Zhang X D, Guo Y Y, Ji Y H, et al. Cytokine release syndrome after modified CAR-NK therapy in an advanced non-small cell lung cancer patient: A case report [J]. Cell Transplantation, 2022, 31: 09636897221094244.
[169] Dhakal B, Berdeja J G, Gregory T, et al. Interim phase I clinical data of FT576 as monotherapy and in combination with daratumumab in subjects with relapsed/refractory multiple myeloma [J]. Blood, 2022, 140(Supplement 1): 4586-4587.
[170] Shi H, Lin Y N, Ran J, et al. Abstract CT099: First-in-human study of ALF501, polypeptide PSMA-targeted chimeric antigen receptor engineered natural killer cells) for castration-resistant prostate cancer [J]. Cancer Research, 2023, 83(8_Supplement): CT099.
[171] Li C J, Yang N N, Li H S, et al. Robo1-specific chimeric antigen receptor natural killer cell therapy for pancreatic ductal adenocarcinoma with liver metastasis [J]. Journal of Cancer Research and Therapeutics, 2020, 16(2): 393-396.
[172] Li Q, Wang Y, Lin M, et al. Abstract A014: Phase I clinical trial with PD-1/MUC1 CAR-pNK92 immunotherapy [J]. Cancer Immunology Research, 2019, 7(2_Supplement): A014.
[173] Burger M C, Forster M T, Romanski A, et al. Intracranial injection of natural killer cells engineered with a HER2-targeted chimeric antigen receptor in patients with recurrent glioblastoma [J]. Neuro-Oncology, 2023, 25(11): 2058-2071.
[174] Khoshandam M, Soltaninejad H, Mousazadeh M, et al. Clinical applications of the CRISPR/Cas9 genome-editing system: Delivery options and challenges in precision medicine [J]. Genes & Diseases, 2024, 11(1): 268-282.
[175] Ahi Y S, Bangari D S, Mittal S K. Adenoviral vector immunity: Its implications and circumvention strategies [J]. Current Gene Therapy, 2011, 11(4): 307-320.
[176] Lino C A, Harper J C, Carney J P, et al. Delivering CRISPR: A review of the challenges and approaches [J]. Drug Delivery, 2018, 25(1): 1234-1257.
[177] Charlesworth C T, Deshpande P S, Dever D P, et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans [J]. Nature Medicine, 2019, 25(2): 249-254.
[178] Fu Y F, Foden J A, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells [J]. Nature Biotechnology, 2013, 31(9): 822-826.
[179] Doench J G, Fusi N, Sullender M, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 [J]. Nature Biotechnology, 2016, 34(2): 184-191.
[180] Kleinstiver B P, Pattanayak V, Prew M S, et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects [J]. Nature, 2016, 529(7587): 490-495.
[181] Leibowitz M L, Papathanasiou S, Doerfler P A, et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing [J]. Nature Genetics, 2021, 53(6): 895-905.
[182] Stadtmauer E A, Fraietta J A, Davis M M, et al. CRISPR-engineered T cells in patients with refractory cancer [J]. Science, 2020, 367(6481): eaba7365.
[183] Cullot G, Boutin J, Toutain J, et al. CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations [J]. Nature Communications, 2019, 10: 1136.
[184] Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements [J]. Nature Biotechnology, 2018, 36(8): 765-771.
[185] Doss M X, Sachinidis A. Current challenges of iPSC-based disease modeling and therapeutic implications [J]. Cells, 2019, 8(5): 403.
[186] Yoshihara M, Hayashizaki Y, Murakawa Y. Genomic instability of iPSCs: Challenges towards their clinical applications [J]. Stem Cell Reviews and Reports, 2017, 13(1): 7-16.
[187] Buckberry S, Liu X D, Poppe D, et al. Transient naive reprogramming corrects hiPS cells functionally and epigenetically [J]. Nature, 2023, 620(7975): 863-872.
[188] Gore A, Li Z, Fung H L, et al. Somatic coding mutations in human induced pluripotent stem cells [J]. Nature, 2011, 471(7336): 63-67.
[189] Dashtban M, Panchalingam K M, Shafa M, et al. Addressing manufacturing challenges for commercialization of iPSC-based therapies [M]//Stem cells and good manufacturing practices. New York: Springer, 2020: 179-198.
[190] Hermanson D L, Ni Z Y, Kaufman D S. Human pluripotent stem cells as a renewable source of natural killer cells [M]//Hematopoietic differentiation of human pluripotent stem cells. Dordrecht: Springer, 2015: 69-79.
[191] Zhu H, Kaufman D S. An improved method to produce clinical-scale natural killer cells from human pluripotent stem cells [M]//In vitro differentiation of T-cells. New York: Springer, 2019: 107-119.
[192] Koehl U, Kalberer C, Spanholtz J, et al. Advances in clinical NK cell studies: Donor selection, manufacturing and quality control [J]. OncoImmunology, 2016, 5(4): e1115178.
[193] Laskowski T J, Biederstädt A, Rezvani K. Natural killer cells in antitumour adoptive cell immunotherapy [J]. Nature Reviews Cancer, 2022, 22(10): 557-575.
[194] Miller J S, Soignier Y, Panoskaltsis-Mortari A, et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer [J]. Blood, 2005, 105(8): 3051-3057.
[195] Agarwal S, Weidner T, Thalheimer F B, et al. In vivo generated human CAR T cells eradicate tumor cells [J]. OncoImmunology, 2019, 8(12): e1671761.
[196] Xu J, Liu L, Parone P, et al. In-vivo B-cell maturation antigen CAR T-cell therapy for relapsed or refractory multiple myeloma [J]. The Lancet, 2025, 406(10500): 228-231.
[197] Bot A, Scharenberg A, Friedman K, et al. In vivo chimeric antigen receptor (CAR)-T cell therapy [J]. Nature Reviews Drug Discovery, 2026, 25(2): 116-137.
[198] Garrison B, Deng H, Yucel G, et al. Senti-202, a selective, off-the-shelf, preclinical CAR-NK cell therapy with CD33 and/or FLT3 activating CAR, healthy cell protection from endomucin (EMCN) inhibitory CAR and calibrated release IL-15 for hematologic malignancies including AML [J]. Blood, 2022, 140(Supplement 1): 4531-4532.

Outlines

/