{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,24]],"date-time":"2026-07-24T09:51:37Z","timestamp":1784886697672,"version":"3.55.0"},"reference-count":79,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T00:00:00Z","timestamp":1765324800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100009704","name":"Fondazione Ri.MED","doi-asserted-by":"crossref","award":["10019"],"award-info":[{"award-number":["10019"]}],"id":[{"id":"10.13039\/501100009704","id-type":"DOI","asserted-by":"crossref"}]},{"name":"ISMETT","award":["I00000219"],"award-info":[{"award-number":["I00000219"]}]},{"name":"Italian Ministry of Health, Ricerca Corrente"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Chimeric antigen receptor (CAR)-modified natural killer (NK) cells represent a promising immunotherapeutic approach for the treatment of oncological malignancies such as hepatocellular carcinoma (HCC). In this work, we have engineered primary human NK cells, re-directing them so they can specifically recognize Glypican-3 (GPC3), an immunotherapeutic target for HCC. In previous studies, we have demonstrated that IFN-\u03b1 significantly enhances NK cells\u2019 anti-tumor and anti-viral cytotoxicity. Fourth-generation self-inactivating lentiviral vectors were used to deliver a transgenic expression of IFN-\u03b1 or its co-expression with IL-15 (which induces NK cells expansion, survival, and function), aiming to enhance CAR-GPC3 NK cells\u2019 anti-tumor response against HCC. We optimized a protocol for efficient transduction of primary NK cells, demonstrating that CAR expression is maintained at high levels over time. Exposure of HCC ectopically expressing GPC3+ to CAR-GPC3-IL15 and CAR-GPC3-IL15-IFN\u03b1 NK cells demonstrated significant in vitro cytotoxicity and cytokine production, dependent on GPC3 expression. To prevent undesired side effects of CAR-NK cell immunotherapy, co-delivery with a suicide gene is advised as a safety measure. Thus, a truncated epidermal growth factor receptor (tEGFR) was co-delivered with the anti-GPC3 CAR, which efficiently promoted the suicide of the CAR-NK used in this work. Our study demonstrates the efficacy of re-directed CAR-GPC3 primary NK cells, encouraging further preclinical and clinical translation studies and strengthening the potential of these cells as a novel treatment option for patients with HCC.<\/jats:p>","DOI":"10.3390\/ijms262411892","type":"journal-article","created":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T12:20:57Z","timestamp":1765369257000},"page":"11892","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Glypican-3-Specific CAR NK Cells Co-Secreting IL-15 and IFN-\u03b1 Have Increased Anti-Tumor Function Versus Hepatocellular Carcinoma In Vitro"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7546-7209","authenticated-orcid":false,"given":"Rosalia","family":"Bus\u00e0","sequence":"first","affiliation":[{"name":"IRCCS ISMETT, 90127 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7710-4735","authenticated-orcid":false,"given":"Gioacchin","family":"Iannolo","sequence":"additional","affiliation":[{"name":"IRCCS ISMETT, 90127 Palermo, Italy"},{"name":"Department of Medicine and Surgery, Kore University of Enna, 94100 Enna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9980-4505","authenticated-orcid":false,"given":"Bruno","family":"Douradinha","sequence":"additional","affiliation":[{"name":"Fondazione Ri.MED, 90133 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3987-9262","authenticated-orcid":false,"given":"Duilio","family":"Pagano","sequence":"additional","affiliation":[{"name":"IRCCS ISMETT, 90127 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Gallina","sequence":"additional","affiliation":[{"name":"IRCCS ISMETT, 90127 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giancarlo","family":"Cappello","sequence":"additional","affiliation":[{"name":"IRCCS ISMETT, 90127 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonio","family":"La Rocca","sequence":"additional","affiliation":[{"name":"IRCCS ISMETT, 90127 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9684-8035","authenticated-orcid":false,"given":"Salvatore","family":"Gruttadauria","sequence":"additional","affiliation":[{"name":"IRCCS ISMETT, 90127 Palermo, Italy"},{"name":"Department of Surgery and Medical and Surgical Specialties, University of Catania, 95124 Catania, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1994-8005","authenticated-orcid":false,"given":"Pier Giulio","family":"Conaldi","sequence":"additional","affiliation":[{"name":"IRCCS ISMETT, 90127 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0154-6270","authenticated-orcid":false,"given":"Ester","family":"Badami","sequence":"additional","affiliation":[{"name":"Fondazione Ri.MED, 90133 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Chakraborty, E., and Sarkar, D. 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