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The disease resists standard chemotherapy and often relapses. Chimeric antigen receptor (CAR) T cell therapy has shown promise for hematological malignancies but faces challenges in solid tumors due to resistance mechanisms like PD-L1 expression, which tumors use to evade the immune system. To address this challenge, we developed fifth-generation CAR T cells targeting integrin \u03b1v\u03b26 that also secrete anti-PD-L1 single-chain variable fragment (scFv) to target both tumor cells and the PD-1\/PD-L1 pathway. We examined integrin \u03b1v\u03b26 and PD-L1 expression in CCA cell lines and engineered T cells to express either fourth-generation CAR T cells targeting integrin \u03b1v\u03b26 (A20 CAR4 T cells) or fifth-generation CAR T cells with anti-PD-L1 scFv secretion (A20 CAR5 T cells). In vitro, A20 CAR5 T cells exhibited less exhaustion and superior long-term functionality compared to A20 CAR4 T cells. In 3D spheroid models of CCA, A20 CAR5 T cells demonstrated enhanced antitumor activity and better infiltration into the spheroid core. These findings suggest that A20 CAR5 T cells have significant potential and warrant further in vivo studies and clinical trials.<\/jats:p>","DOI":"10.1186\/s12967-025-06453-y","type":"journal-article","created":{"date-parts":[[2025,4,16]],"date-time":"2025-04-16T14:18:52Z","timestamp":1744813132000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Enhanced cytotoxicity against cholangiocarcinoma by fifth-generation chimeric antigen receptor T cells targeting integrin \u03b1v\u03b26 and secreting anti-PD-L1 scFv"],"prefix":"10.1186","volume":"23","author":[{"given":"Nattaporn","family":"Phanthaphol","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chalermchai","family":"Somboonpatarakun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kwanpirom","family":"Suwanchiwasiri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pornpimon","family":"Yuti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jatuporn","family":"Sujjitjoon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Punn","family":"Augsornworawat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"George S.","family":"Baillie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8347-3000","authenticated-orcid":false,"given":"Mutita","family":"Junking","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pa-thai","family":"Yenchitsomanus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,4,16]]},"reference":[{"issue":"9","key":"6453_CR1","doi-asserted-by":"publisher","first-page":"557","DOI":"10.1038\/s41575-020-0310-z","volume":"17","author":"JM Banales","year":"2020","unstructured":"Banales JM, et al. 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