{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T02:50:22Z","timestamp":1785898222624,"version":"3.56.0"},"reference-count":39,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2024,1,12]],"date-time":"2024-01-12T00:00:00Z","timestamp":1705017600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"iPRIME Else-Kr\u00f6ner-Promotionskollegium"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>Background: CAR-T cell therapy has shown impressive results and is now part of standard-of-care treatment of B-lineage malignancies, whereas the treatment of myeloid diseases has been limited by the lack of suitable targets. CD45 is expressed on almost all types of blood cells including myeloid leukemia cells, but not on non-hematopoietic tissue, making it a potential target for CAR-directed therapy. Because of its high expression on T and NK cells, fratricide is expected to hinder CD45CAR-mediated therapy. Due to its important roles in effector cell activation, signal transduction and cytotoxicity, CD45 knockout aimed at preventing fratricide in T and NK cells has been expected to lead to considerable functional impairment. Methods: CD45 knockout was established on T and NK cell lines using CRISPR\/Cas9-RNPs and electroporation, and the successful protocol was transferred to primary T cells. A combined protocol was developed enabling CD45 knockout and retroviral transduction with a third-generation CAR targeting CD45 or CD19. The functionality of CD45ko effector cells, CD45ko\/CD45CAR-T and CD45ko\/CD19CAR-T cells was studied using proliferation as well as short- and long-term cytotoxicity assays. Results: As expected, the introduction of a CD45-CAR into T cells resulted in potent fratricide that can be avoided by CD45 knockout. Unexpectedly, the latter had no negative impact on T- and NK-cell proliferation in vitro. Moreover, CD45ko\/CD45CAR-T cells showed potent cytotoxicity against CD45-expressing AML and lymphoma cell lines in short-term and long-term co-culture assays. A pronounced cytotoxicity of CD45ko\/CD45CAR-T cells was maintained even after four weeks of culture. In a further setup, we confirmed the conserved functionality of CD45ko cells using a CD19-CAR. Again, the proliferation and cytotoxicity of CD45ko\/CD19CAR-T cells showed no differences from those of their CD45-positive counterparts in vitro. Conclusions: We report the efficient production of highly and durably active CD45ko\/CAR-T cells. CD45 knockout did not impair the functionality of CAR-T cells in vitro, irrespective of the target antigen. If their activity can be confirmed in vivo, CD45ko\/CD45CAR-T cells might, for example, be useful as part of conditioning regimens prior to stem cell transplantation.<\/jats:p>","DOI":"10.3390\/cancers16020334","type":"journal-article","created":{"date-parts":[[2024,1,12]],"date-time":"2024-01-12T03:54:34Z","timestamp":1705031674000},"page":"334","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["CD45-Directed CAR-T Cells with CD45 Knockout Efficiently Kill Myeloid Leukemia and Lymphoma Cells In Vitro Even after Extended Culture"],"prefix":"10.3390","volume":"16","author":[{"given":"Maraike","family":"Harfmann","sequence":"first","affiliation":[{"name":"Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tanja","family":"Schr\u00f6der","sequence":"additional","affiliation":[{"name":"Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dawid","family":"G\u0142\u00f3w","sequence":"additional","affiliation":[{"name":"Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maximilian","family":"Jung","sequence":"additional","affiliation":[{"name":"Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Almut","family":"Uhde","sequence":"additional","affiliation":[{"name":"Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicolaus","family":"Kr\u00f6ger","sequence":"additional","affiliation":[{"name":"Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefan","family":"Horn","sequence":"additional","affiliation":[{"name":"Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9050-6766","authenticated-orcid":false,"given":"Kristoffer","family":"Riecken","sequence":"additional","affiliation":[{"name":"Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9780-7211","authenticated-orcid":false,"given":"Boris","family":"Fehse","sequence":"additional","affiliation":[{"name":"Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francis A.","family":"Ayuk","sequence":"additional","affiliation":[{"name":"Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,1,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1016\/S0140-6736(21)00933-8","article-title":"Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy in patients with relapsed or refractory multiple myeloma (CARTITUDE-1): A phase 1b\/2 open-label study","volume":"398","author":"Berdeja","year":"2021","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1056\/NEJMoa2024850","article-title":"Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma","volume":"384","author":"Munshi","year":"2021","journal-title":"N. 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