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However, comparable efficacy in solid tumors remains limited, partly due to poor CAR T cell persistence and immune-mediated rejection. A major contributor, which has hampered the clinical efficacy of CAR T cells in clinical practice, is the immunogenicity of the murine-derived single-chain variable fragments (scFvs) commonly used in CAR constructs. Cell and humoral immune responses to the murine parts of CARs have been implicated in CAR T cell rejection. Here, we describe the generation and in vitro characterization of humanized CAR T cells targeting prostate-specific membrane antigen (PSMA) on prostate cancer cells, based on two distinct murine scFvs (A5 and D7). Humanization improved the germinality index and successfully preserved CAR surface expression. Functional assays demonstrated that humanized PSMA-CAR T cells retained antigen-specific binding, activation and cytotoxicity, differentiation, exhaustion and cytokine secretion profiles comparable to their murine counterparts. These results support the feasibility of humanization as a strategy to reduce immunogenicity without compromising CAR T cell capabilities, providing a foundation for further in vivo validation in solid tumor settings.<\/jats:p>","DOI":"10.3390\/cells14171333","type":"journal-article","created":{"date-parts":[[2025,8,28]],"date-time":"2025-08-28T15:03:14Z","timestamp":1756393394000},"page":"1333","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Engineering of Humanized PSMA-Directed CAR T Cells for Potent and Specific Elimination of Prostate Cancer Cells"],"prefix":"10.3390","volume":"14","author":[{"given":"Tobias D.","family":"Deller","sequence":"first","affiliation":[{"name":"Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany"},{"name":"Center for Cell and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany"},{"name":"Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4284-2006","authenticated-orcid":false,"given":"Jamal","family":"Alzubi","sequence":"additional","affiliation":[{"name":"Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany"},{"name":"Center for Cell and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany"},{"name":"Department of Haematooncology, Faculty of Medicine, University of Ostrava, 70852 Ostrava, Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8877-8359","authenticated-orcid":false,"given":"Laura","family":"Mosti","sequence":"additional","affiliation":[{"name":"Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-7951-3205","authenticated-orcid":false,"given":"Marie","family":"Peschers","sequence":"additional","affiliation":[{"name":"Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany"},{"name":"Department of Urology, Medical Center-University of Freiburg, 79106 Freiburg, Germany"},{"name":"Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Gratzke","sequence":"additional","affiliation":[{"name":"Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany"},{"name":"Department of Urology, Medical Center-University of Freiburg, 79106 Freiburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6779-4325","authenticated-orcid":false,"given":"Philipp","family":"Wolf","sequence":"additional","affiliation":[{"name":"Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany"},{"name":"Department of Urology, Medical Center-University of Freiburg, 79106 Freiburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7757-4630","authenticated-orcid":false,"given":"Toni","family":"Cathomen","sequence":"additional","affiliation":[{"name":"Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany"},{"name":"Center for Cell and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany"},{"name":"Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Wittibschlager, V., Bacher, U., Seipel, K., Porret, N., Wiedemann, G., Haslebacher, C., Hoffmann, M., Daskalakis, M., Akhoundova, D., and Pabst, T. 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