{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T19:00:15Z","timestamp":1790794815926,"version":"4.1.0"},"reference-count":44,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2022,10,24]],"date-time":"2022-10-24T00:00:00Z","timestamp":1666569600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Stefan Morsch Stiftung, Clinician Scientist Program","award":["440-0-0"],"award-info":[{"award-number":["440-0-0"]}]},{"name":"MaxCyte Inc.","award":["440-0-0"],"award-info":[{"award-number":["440-0-0"]}]},{"name":"University Children\u2019s Hospital of T\u00fcbingen","award":["440-0-0"],"award-info":[{"award-number":["440-0-0"]}]},{"name":"Open Access Publishing Fund of University of T\u00fcbingen","award":["440-0-0"],"award-info":[{"award-number":["440-0-0"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Acute myeloid leukemia (AML) and B-cell acute lymphocytic leukemia (B-ALL) are severe blood malignancies affecting both adults and children. Chimeric antigen receptor (CAR)-based immunotherapies have proven highly efficacious in the treatment of leukemia. However, the challenge of the immune escape of cancer cells remains. The development of more affordable and ready-to-use therapies is essential in view of the costly and time-consuming preparation of primary cell-based treatments. In order to promote the antitumor function against AML and B-ALL, we transduced NK-92 cells with CD276-CAR or CD19-CAR constructs. We also attempted to enhance cytotoxicity by a gene knockout of three different inhibitory checkpoints in NK cell function (CBLB, NKG2A, TIGIT) with CRISPR-Cas9 technology. The antileukemic activity of the generated cell lines was tested with calcein and luciferase-based cytotoxicity assays in various leukemia cell lines. Both CAR-NK-92 exhibited targeted cytotoxicity and a significant boost in antileukemic function in comparison to parental NK-92. CRISPR-Cas9 knock-outs did not improve B-ALL cytotoxicity. However, triple knock-out CD276-CAR-NK-92 cells, as well as CBLB or TIGIT knock-out NK-92 cells, showed significantly enhanced cytotoxicity against U-937 or U-937 CD19\/tag AML cell lines. These results indicate that the CD19-CAR and CD276-CAR-NK-92 cell lines\u2019 cytotoxic performance is suitable for leukemia killing, making them promising off-the-shelf therapeutic candidates. The knock-out of CBLB and TIGIT in NK-92 and CD276-CAR-NK-92 should be further investigated for the treatment of AML.<\/jats:p>","DOI":"10.3390\/ijms232112828","type":"journal-article","created":{"date-parts":[[2022,10,24]],"date-time":"2022-10-24T11:53:55Z","timestamp":1666612435000},"page":"12828","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":49,"title":["Preclinical Evaluation of CRISPR-Edited CAR-NK-92 Cells for Off-the-Shelf Treatment of AML and B-ALL"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4699-7486","authenticated-orcid":false,"given":"Guillermo","family":"Ure\u00f1a-Bail\u00e9n","sequence":"first","affiliation":[{"name":"Department of Hematology and Oncology, Children\u2019s Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J\u00e9r\u00f4me-Maurice","family":"Dobrowolski","sequence":"additional","affiliation":[{"name":"Department of Hematology and Oncology, Children\u2019s Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yujuan","family":"Hou","sequence":"additional","affiliation":[{"name":"Department of Hematology and Oncology, Children\u2019s Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alicia","family":"Dirlam","sequence":"additional","affiliation":[{"name":"Department of Hematology and Oncology, Children\u2019s Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0775-5820","authenticated-orcid":false,"given":"Alicia","family":"Roig-Merino","sequence":"additional","affiliation":[{"name":"MaxCyte Inc., Rockville, MD 20850, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabine","family":"Schleicher","sequence":"additional","affiliation":[{"name":"Department of Hematology and Oncology, Children\u2019s Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Atar","sequence":"additional","affiliation":[{"name":"Department of Hematology and Oncology, Children\u2019s Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0520-7866","authenticated-orcid":false,"given":"Christian","family":"Seitz","sequence":"additional","affiliation":[{"name":"Department of Hematology and Oncology, Children\u2019s Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany"},{"name":"Cluster of Excellence iFIT (EXC2180) \u201cImage-Guided and Functionally Instructed Tumor Therapies\u201d, University of T\u00fcbingen, 72074 Tuebingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Judith","family":"Feucht","sequence":"additional","affiliation":[{"name":"Department of Hematology and Oncology, Children\u2019s Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany"},{"name":"Cluster of Excellence iFIT (EXC2180) \u201cImage-Guided and Functionally Instructed Tumor Therapies\u201d, University of T\u00fcbingen, 72074 Tuebingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Justin S.","family":"Antony","sequence":"additional","affiliation":[{"name":"Department of Hematology and Oncology, Children\u2019s Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tahereh","family":"Mohammadian Gol","sequence":"additional","affiliation":[{"name":"Department of Hematology and Oncology, Children\u2019s Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rupert","family":"Handgretinger","sequence":"additional","affiliation":[{"name":"Department of Hematology and Oncology, Children\u2019s Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0211-2343","authenticated-orcid":false,"given":"Markus","family":"Mezger","sequence":"additional","affiliation":[{"name":"Department of Hematology and Oncology, Children\u2019s Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,10,24]]},"reference":[{"key":"ref_1","first-page":"731","article-title":"Leukemia: An overview for primary care","volume":"89","author":"Davis","year":"2014","journal-title":"Am. 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