{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,7]],"date-time":"2026-11-07T04:52:13Z","timestamp":1794027133294,"version":"4.8.0"},"reference-count":39,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2025,9,5]],"date-time":"2025-09-05T00:00:00Z","timestamp":1757030400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Intramural Research Program of the Center for Biologics Evaluation and Research (CBER), U.S. Food and Drug Administration"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>Chimeric antigen receptor-expressing NK (CAR-NK) cells represent an advancing frontier in cancer immunotherapy, building upon decades of natural killer cell research and recent breakthroughs in CAR technology. While early CAR-NK manufacturing protocols have demonstrated feasibility, existing manufacturing methods, whether utilizing cord blood or peripheral blood sources, often require extended culture periods and intensive labor, creating bottlenecks for widespread therapeutic application. To address these manufacturing hurdles, we have developed an optimized protocol for ex vivo CAR-NK cell production from human peripheral blood that incorporates lessons learned from previous methodologies while introducing novel efficiency improvements. This protocol offers a practical solution for scalable CAR-NK cell manufacturing that can be readily adapted across different production facilities, potentially accelerating the clinical development of CAR-NK therapies.<\/jats:p>","DOI":"10.3390\/mps8050102","type":"journal-article","created":{"date-parts":[[2025,9,5]],"date-time":"2025-09-05T09:51:21Z","timestamp":1757065881000},"page":"102","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["A Scalable Protocol for Ex Vivo Production of CAR-Engineered Human NK Cells"],"prefix":"10.3390","volume":"8","author":[{"given":"Supreet","family":"Khanal","sequence":"first","affiliation":[{"name":"Tumor Vaccine and Biotechnology Branch, Division of Cell Therapy II, Office of Cellular Therapy and Human Tissues, Office of Therapeutic Products, Center for Biologics Evaluation and Research, U.S. FDA, Silver Spring, MD 20993, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nirjal","family":"Bhattarai","sequence":"additional","affiliation":[{"name":"Tumor Vaccine and Biotechnology Branch, Division of Cell Therapy II, Office of Cellular Therapy and Human Tissues, Office of Therapeutic Products, Center for Biologics Evaluation and Research, U.S. FDA, Silver Spring, MD 20993, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"545","DOI":"10.1056\/NEJMoa1910607","article-title":"Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors","volume":"382","author":"Liu","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Rezvani, K., and Rouce, R.H. (2015). The application of natural killer cell immunotherapy for the treatment of cancer. Front. Immunol., 6.","DOI":"10.3389\/fimmu.2015.00578"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"16015","DOI":"10.1038\/mto.2016.15","article-title":"Clinical manufacturing of CAR T cells: Foundation of a promising therapy","volume":"3","author":"Wang","year":"2016","journal-title":"Mol. Ther.-Oncolytics"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1038\/s41573-019-0052-1","article-title":"NK cells for cancer immunotherapy","volume":"19","author":"Shimasaki","year":"2020","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Xie, G., Dong, H., Liang, Y., Ham, J.D., Rizwan, R., and Chen, J. (2020). CAR-NK cells: A promising cellular immunotherapy for cancer. eBioMedicine, 59.","DOI":"10.1016\/j.ebiom.2020.102975"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1038\/s41586-023-06945-1","article-title":"Natural killer cell therapies","volume":"626","author":"Vivier","year":"2024","journal-title":"Nature"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Wang, X., Byrne, M.E., Liu, C., Ma, M.T., and Liu, D. (2024). Scalable process development of NK and CAR-NK expansion in a closed bioreactor. Front. Immunol., 15.","DOI":"10.3389\/fimmu.2024.1412378"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1038\/s41420-024-02077-1","article-title":"Emerging roles of CAR-NK cell therapies in tumor immunotherapy: Current status and future directions","volume":"10","author":"Zhong","year":"2024","journal-title":"Cell Death Discov."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Khanal, S., Baer, A., Hossain, M.K., Colon-Moran, W., Panthi, S., and Bhattarai, N. (2024). Soluble factors released by peripheral blood-derived CAR-NK cells cause bystander myeloid cell activation. Front. Immunol., 15.","DOI":"10.3389\/fimmu.2024.1519415"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Tarannum, M., Romee, R., and Shapiro, R.M. (2022). Innovative strategies to improve the clinical application of NK cell-based immunotherapy. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.859177"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1038\/s41591-023-02785-8","article-title":"Safety, efficacy and determinants of response of allogeneic CD19-specific CAR-NK cells in CD19+ B cell tumors: A phase 1\/2 trial","volume":"30","author":"Marin","year":"2024","journal-title":"Nat. Med."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1007\/s12032-024-02362-0","article-title":"A new vision of the efficacy of both CAR-NK and CAR-T cells in treating cancers and autoimmune diseases","volume":"41","author":"Hassan","year":"2024","journal-title":"Med. Oncol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Yu, J., Yang, Y., Gu, Z., Shi, M., La Cava, A., and Liu, A. (2024). CAR immunotherapy in autoimmune diseases: Promises and challenges. Front. Immunol., 15.","DOI":"10.3389\/fimmu.2024.1461102"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"W\u0142odarczyk, M., and Pyrzynska, B. (2022). CAR-NK as a rapidly developed and efficient immunotherapeutic strategy against cancer. Cancers, 15.","DOI":"10.3390\/cancers15010117"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1013","DOI":"10.1016\/j.jcyt.2025.05.008","article-title":"Feeder cell-free production of CAR-NK cells via activation of PKC and calcium signaling pathways","volume":"27","author":"Khanal","year":"2025","journal-title":"Cytotherapy"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1038\/s41409-023-02180-4","article-title":"Manufacturing of primary CAR-NK cells in an automated system for the treatment of acute myeloid leukemia","volume":"59","author":"Albinger","year":"2024","journal-title":"Bone Marrow Transplant."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/j.reth.2023.05.006","article-title":"An efficient feeder-free and chemically-defined expansion strategy for highly purified natural killer cells derived from human cord blood","volume":"24","author":"Nakazawa","year":"2023","journal-title":"Regen. Ther."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1853314","DOI":"10.1080\/2162402X.2020.1853314","article-title":"Expanded NK cells from umbilical cord blood and adult peripheral blood combined with daratumumab are effective against tumor cells from multiple myeloma patients","volume":"10","author":"Constantinides","year":"2021","journal-title":"Oncoimmunology"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"408","DOI":"10.1158\/2643-3230.BCD-21-0084","article-title":"Scalable manufacturing of CAR T cells for cancer immunotherapy","volume":"2","author":"Elsallab","year":"2021","journal-title":"Blood Cancer Discov."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Johnson, C.D., Zale, N.E., Frary, E.D., and Lomakin, J.A. (2022). Feeder-cell-free and serum-free expansion of natural killer cells using Cloudz microspheres, G-Rex6M, and human platelet lysate. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.803380"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Ojo, E.O., Sharma, A.A., Liu, R., Moreton, S., Checkley-Luttge, M.A., Gupta, K., Lee, G., Lee, D.A., Otegbeye, F., and Sekaly, R.P. (2019). Membrane bound IL-21 based NK cell feeder cells drive robust expansion and metabolic activation of NK cells. Sci. Rep., 9.","DOI":"10.1038\/s41598-019-51287-6"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4010","DOI":"10.1158\/0008-5472.CAN-08-3712","article-title":"Expansion of highly cytotoxic human natural killer cells for cancer cell therapy","volume":"69","author":"Fujisaki","year":"2009","journal-title":"Cancer Res."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Heinze, A., Grebe, B., Bremm, M., Huenecke, S., Munir, T.A., Graafen, L., Frueh, J.T., Merker, M., Rettinger, E., and Soerensen, J. (2019). 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. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.02816"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e96219","DOI":"10.1172\/jci.insight.96219","article-title":"Continuous treatment with IL-15 exhausts human NK cells via a metabolic defect","volume":"3","author":"Felices","year":"2018","journal-title":"JCI Insight"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"876","DOI":"10.1016\/j.imbio.2015.01.009","article-title":"Multiple effects of IL-21 on human NK cells in ex vivo expansion","volume":"220","author":"Li","year":"2015","journal-title":"Immunobiology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1038\/s41420-024-01815-9","article-title":"Breakthrough of solid tumor treatment: CAR-NK immunotherapy","volume":"10","author":"Wang","year":"2024","journal-title":"Cell Death Discov."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e1507","DOI":"10.1002\/cti2.1507","article-title":"Optimisation of a primary human CAR-NK cell manufacturing pipeline","volume":"13","author":"Pfefferle","year":"2024","journal-title":"Clin. Transl. Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1571","DOI":"10.1038\/s41551-024-01219-1","article-title":"A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells","volume":"8","author":"Sin","year":"2024","journal-title":"Nat. Biomed. Eng."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"6824","DOI":"10.1182\/blood-2023-178289","article-title":"CD19-specific CAR NK cells coexpressing IL-21 exhibit superior expansion and antitumor activity against CD19-postive lymphoma","volume":"142","author":"He","year":"2023","journal-title":"Blood"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"ltac003","DOI":"10.1093\/immadv\/ltac003","article-title":"Engineering CAR-NK cells: How to tune innate killer cells for cancer immunotherapy","volume":"2","author":"Schmidt","year":"2022","journal-title":"Immunother. Adv."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1183","DOI":"10.15252\/emmm.201607485","article-title":"Clinical development of CAR T cells-challenges and opportunities in translating innovative treatment concepts","volume":"9","author":"Hartmann","year":"2017","journal-title":"EMBO Mol. Med."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1871","DOI":"10.1016\/j.xphs.2020.12.015","article-title":"Addressing Patient to Patient Variability for Autologous CAR T Therapies","volume":"110","author":"Jiang","year":"2021","journal-title":"J. Pharm. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1100","DOI":"10.1158\/2326-6066.CIR-17-0405","article-title":"Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells","volume":"6","author":"Ghassemi","year":"2018","journal-title":"Cancer Immunol. Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1002","DOI":"10.1016\/j.jcyt.2016.05.009","article-title":"Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy","volume":"18","author":"Mock","year":"2016","journal-title":"Cytotherapy"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Watanabe, N., Mo, F., and McKenna, M.K. (2022). Impact of Manufacturing Procedures on CAR T Cell Functionality. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.876339"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1172\/JCI32103","article-title":"Adoptive transfer of effector CD8+ T cells derived from central memory cells establishes persistent T cell memory in primates","volume":"118","author":"Berger","year":"2008","journal-title":"J. Clin. Investig."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.omtm.2016.12.006","article-title":"Global Manufacturing of CAR T Cell Therapy","volume":"4","author":"Levine","year":"2017","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1016\/j.jcyt.2019.01.003","article-title":"Chimeric antigen receptor-T cell therapy manufacturing: Modelling the effect of offshore production on aggregate cost of goods","volume":"21","author":"Harrison","year":"2019","journal-title":"Cytotherapy"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"786","DOI":"10.1038\/s43018-025-00940-3","article-title":"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","volume":"6","author":"Lei","year":"2025","journal-title":"Nat. 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