{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:47:34Z","timestamp":1785415654583,"version":"3.56.0"},"posted":{"date-parts":[[2026,7,5]]},"group-title":"Immunology","reference-count":32,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,5]],"date-time":"2026-07-05T00:00:00Z","timestamp":1783209600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"award":["117854"],"award-info":[{"award-number":["117854"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"award":["ZIA BC 011855"],"award-info":[{"award-number":["ZIA BC 011855"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"award":["HHSN261201500003I"],"award-info":[{"award-number":["HHSN261201500003I"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"crossref","award":["Project-ID 546185154"],"award-info":[{"award-number":["Project-ID 546185154"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,5]]},"abstract":"Abstract<\/jats:title>\n \n Background & Aims<\/jats:title>\n \n Liver transplantation improves outcomes in hepatocellular carcinoma (HCC), yet treatment options for patients with tumor recurrence remain limited to tyrosine kinase inhibitors. Glypican-3 (GPC3)-targeted CAR T cells offer a tumor-directed immune-based therapeutic strategy, but their efficacy may be limited by post-transplant immunosuppression. We developed a CAR T cell platform combining CRISPR\/Cas9-mediated\n FKBP1A<\/jats:italic>\n disruption to confer resistance to FKBP12-dependent immunosuppressive agents, including tacrolimus, everolimus, and sirolimus, with\n TRAC<\/jats:italic>\n knockout to eliminate endogenous T cell receptor expression and reduce alloreactivity.\n <\/jats:p>\n <\/jats:sec>\n \n Methods<\/jats:title>\n \n Human T cells were edited using Cas9 ribonucleoprotein complexes targeting\n FKBP1A<\/jats:italic>\n and\n TRAC<\/jats:italic>\n , expanded, and transduced with an anti-GPC3 CAR construct. Cytokine production and cytotoxicity were assessed\n in vitro<\/jats:italic>\n . Antitumor activity under tacrolimus treatment was evaluated in a Hep G2 xenograft model, and xenoreactivity was assessed in a graft-versus-host disease model.\n FKBP1A\/TRAC<\/jats:italic>\n double-knockout T cells were enriched using mTOR inhibitor selection combined with CD3-based MACS depletion. PBMCs from liver transplant recipients were used to evaluate feasibility for clinical translation during the early post-transplant period.\n <\/jats:p>\n <\/jats:sec>\n \n Results<\/jats:title>\n \n Tacrolimus suppressed wild-type CAR T cell function but not\n FKBP1A<\/jats:italic>\n \/\n TRAC<\/jats:italic>\n double-knockout CAR T cells, which retained cytokine production, cytotoxicity, and\n in vivo<\/jats:italic>\n antitumor activity. Cyclosporine A remained suppressive, enabling its potential use as a pharmacologic control strategy.\n TRAC<\/jats:italic>\n disruption reduced xenoreactivity. CD3-based MACS depletion and mTOR inhibition achieved functional double-knockout efficiencies greater than 98%, without compromising cell viability. Functional\n FKBP1A\/TRAC<\/jats:italic>\n knockout CAR T cells were generated from patient PBMC samples 30 days post-transplant.\n <\/jats:p>\n <\/jats:sec>\n \n Conclusions<\/jats:title>\n Dual-edited GPC3 CAR T cells resist tacrolimus-based immunosuppression while limiting alloreactivity, supporting their use for recurrent HCC after liver transplantation. Sequential, high-viability selection in a modular cellular engineering framework enables adaptation to alternative tumor targets and next-generation CAR T cell designs.<\/jats:p>\n <\/jats:sec>\n \n Impact and implications<\/jats:title>\n \n \n Engineering armored GPC3-targeting CAR T cells for hepatocellular carcinoma in the post\u2013transplant setting<\/jats:p>\n <\/jats:list-item>\n \n Demonstrating safety and therapeutic efficacy in the presence of immunosuppressive drugs in preclinical models<\/jats:p>\n <\/jats:list-item>\n \n Leveraging mTOR inhibitor resistance and CD3 destabilization enables high-efficiency, high-purity selection of genetically modified T cells<\/jats:p>\n <\/jats:list-item>\n \n A modular cellular engineering platform enables utilization for alternative tumor targets and next-generation CAR T cells<\/jats:p>\n <\/jats:list-item>\n <\/jats:list>\n <\/jats:sec>","DOI":"10.64898\/2026.06.30.735569","type":"posted-content","created":{"date-parts":[[2026,7,6]],"date-time":"2026-07-06T04:15:12Z","timestamp":1783311312000},"source":"Crossref","is-referenced-by-count":0,"title":["Engineering CAR T Cells for Hepatocellular Carcinoma Recurrence after Liver Transplantation"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0036-2234","authenticated-orcid":false,"given":"Lorenz","family":"Kocheise","sequence":"first","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriel","family":"Bacil","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pavan","family":"Bhimalli","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohamed-Reda","family":"Benmebarek","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dan","family":"Li","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick","family":"Huang","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chi","family":"Ma","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vinona","family":"Muralidaran","sequence":"additional","affiliation":[{"name":"MedStar Georgetown Transplant Institute, MedStar Georgetown University Hospital and Center for Translational Transplant Medicine, Georgetown University Medical Center, Washington, DC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jorge","family":"Hernandez-Felix","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph R.","family":"Bugliarelli","sequence":"additional","affiliation":[{"name":"Genome Modification Core, Laboratory Animal Sciences Program, Frederick National Lab for Cancer Research, Frederick, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raj","family":"Chari","sequence":"additional","affiliation":[{"name":"Genome Modification Core, Laboratory Animal Sciences Program, Frederick National Lab for Cancer Research, Frederick, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kylynda","family":"Bauer","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuta","family":"Myojin","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Firdaus","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiao Bin","family":"Zhu","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Corbin","family":"Morris","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Firouzeh","family":"Korangy","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Kroemer","sequence":"additional","affiliation":[{"name":"MedStar Georgetown Transplant Institute, MedStar Georgetown University Hospital and Center for Translational Transplant Medicine, Georgetown University Medical Center, Washington, DC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mitchell","family":"Ho","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"},{"name":"Antibody Engineering Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tim F.","family":"Greten","sequence":"additional","affiliation":[{"name":"Gastrointestinal Malignancies Section, Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"},{"name":"Liver Cancer Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026070805050643000_2026.06.30.735569v1.1","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1200\/JCO.24.00857","article-title":"Liver Transplantation for Hepatocellular Carcinoma: An Expanding Cornerstone of Care in the Era of Immunotherapy","volume":"43","year":"2025","journal-title":"J Clin Oncol"},{"key":"2026070805050643000_2026.06.30.735569v1.2","doi-asserted-by":"crossref","unstructured":"Rezaee-Zavareh MS , Ahn JC , Kim H , et al. Liver transplantation is the major determinant of >\/=10-year survival in patients with hepatocellular carcinoma. Hepatol Commun 2026;10.","DOI":"10.1097\/HC9.0000000000000951"},{"key":"2026070805050643000_2026.06.30.735569v1.3","first-page":"779","article-title":"Ten-Year Outcomes of Liver Transplant and Downstaging for Hepatocellular Carcinoma","volume":"157","year":"2022","journal-title":"JAMA Surg"},{"key":"2026070805050643000_2026.06.30.735569v1.4","doi-asserted-by":"publisher","DOI":"10.1002\/hep.30879"},{"key":"2026070805050643000_2026.06.30.735569v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.jhep.2023.01.030"},{"key":"2026070805050643000_2026.06.30.735569v1.6","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/j.jhep.2026.02.019","article-title":"Efficacy of liver transplantation after response to atezolizumab-bevacizumab downstaging of intermediate and advanced hepatocellular carcinoma (ImmunoXXL)","volume":"85","year":"2026","journal-title":"J Hepatol"},{"key":"2026070805050643000_2026.06.30.735569v1.7","doi-asserted-by":"publisher","DOI":"10.1053\/j.gastro.2017.09.025"},{"key":"2026070805050643000_2026.06.30.735569v1.8","doi-asserted-by":"crossref","unstructured":"Kocheise L , Benmebarek M-R , Ho M , et al. Chimeric antigen receptor therapy for hepatocellular carcinoma. Seminars in Immunology 2026;81.","DOI":"10.1016\/j.smim.2025.102000"},{"key":"2026070805050643000_2026.06.30.735569v1.9","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-08261-8"},{"key":"2026070805050643000_2026.06.30.735569v1.10","doi-asserted-by":"publisher","DOI":"10.1200\/JCO.2024.42.16_suppl.4019"},{"key":"2026070805050643000_2026.06.30.735569v1.11","doi-asserted-by":"publisher","DOI":"10.1053\/j.gastro.2020.02.011"},{"key":"2026070805050643000_2026.06.30.735569v1.12","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gku936"},{"key":"2026070805050643000_2026.06.30.735569v1.13","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2012-01-405365"},{"key":"2026070805050643000_2026.06.30.735569v1.14","doi-asserted-by":"publisher","DOI":"10.1016\/S0016-5085(03)00689-9"},{"key":"2026070805050643000_2026.06.30.735569v1.15","doi-asserted-by":"crossref","first-page":"574","DOI":"10.1002\/hep.29844","article-title":"Identification of alpha-fetoprotein-specific T-cell receptors for hepatocellular carcinoma immunotherapy","volume":"68","year":"2018","journal-title":"Hepatology"},{"key":"2026070805050643000_2026.06.30.735569v1.16","doi-asserted-by":"publisher","DOI":"10.1016\/j.jceh.2022.06.007"},{"key":"2026070805050643000_2026.06.30.735569v1.17","doi-asserted-by":"publisher","DOI":"10.1097\/TP.0000000000000965"},{"key":"2026070805050643000_2026.06.30.735569v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2024.06.022"},{"key":"2026070805050643000_2026.06.30.735569v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2020.09.011"},{"key":"2026070805050643000_2026.06.30.735569v1.20","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-44176-0"},{"key":"2026070805050643000_2026.06.30.735569v1.21","doi-asserted-by":"publisher","DOI":"10.1016\/j.jhep.2025.10.020"},{"key":"2026070805050643000_2026.06.30.735569v1.22","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1002\/hep.31662","article-title":"Immunosuppressive Drug-Resistant Armored T-Cell Receptor T Cells for Immune Therapy of HCC in Liver Transplant Patients","volume":"74","year":"2021","journal-title":"Hepatology"},{"key":"2026070805050643000_2026.06.30.735569v1.23","first-page":"A68","article-title":"109 Dominant-negative TGF\u03b2 receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma","volume":"8","year":"2020","journal-title":"Journal for ImmunoTherapy of Cancer"},{"key":"2026070805050643000_2026.06.30.735569v1.24","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/j.jhep.2025.07.033","article-title":"Phase I trial of ADP-A2AFP TCR T-cell therapy in patients with advanced hepatocellular or gastric hepatoid carcinoma","volume":"84","year":"2026","journal-title":"J Hepatol"},{"key":"2026070805050643000_2026.06.30.735569v1.25","doi-asserted-by":"publisher","DOI":"10.1016\/j.jhep.2014.10.001"},{"key":"2026070805050643000_2026.06.30.735569v1.26","doi-asserted-by":"crossref","first-page":"e2518996122","DOI":"10.1073\/pnas.2518996122","article-title":"Dual-targeted ping-pong CAR T cells: Leveraging peripheral expansion to improve solid tumor immunotherapy","volume":"122","year":"2025","journal-title":"Proc Natl Acad Sci U S A"},{"key":"2026070805050643000_2026.06.30.735569v1.27","doi-asserted-by":"crossref","first-page":"101881","DOI":"10.1016\/j.trim.2023.101881","article-title":"Diagnosis and treatment of adult patients with PTLD at different sites after liver transplantation: A three-case report and literature review","volume":"80","year":"2023","journal-title":"Transpl Immunol"},{"key":"2026070805050643000_2026.06.30.735569v1.28","doi-asserted-by":"crossref","first-page":"2","DOI":"10.21037\/aol-24-14","article-title":"Walking the tightrope: acute liver rejection after chimeric antigen receptor-T cell therapy for post-transplant lymphoproliferative disorder (PTLD)\u2014a case report","volume":"9","year":"2025","journal-title":"Annals of Lymphoma"},{"key":"2026070805050643000_2026.06.30.735569v1.29","doi-asserted-by":"crossref","first-page":"2043","DOI":"10.1038\/s41587-024-02531-6","article-title":"SEED-Selection enables high-efficiency enrichment of primary T cells edited at multiple loci","volume":"43","year":"2025","journal-title":"Nat Biotechnol"},{"key":"2026070805050643000_2026.06.30.735569v1.30","doi-asserted-by":"crossref","first-page":"2150","DOI":"10.1016\/j.cgh.2023.04.005","article-title":"Liver Transplantation 2023: Status Report, Current and Future Challenges","volume":"21","year":"2023","journal-title":"Clin Gastroenterol Hepatol"},{"key":"2026070805050643000_2026.06.30.735569v1.31","doi-asserted-by":"crossref","first-page":"1029","DOI":"10.1038\/s41586-025-08799-1","article-title":"Gene-modified pig-to-human liver xenotransplantation","volume":"641","year":"2025","journal-title":"Nature"},{"key":"2026070805050643000_2026.06.30.735569v1.32","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1016\/j.jhep.2025.08.044","article-title":"Genetically engineered pig-to-human liver xenotransplantation","volume":"84","year":"2026","journal-title":"J Hepatol"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.06.30.735569","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T12:05:17Z","timestamp":1783512317000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.06.30.735569"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,5]]},"references-count":32,"URL":"https:\/\/doi.org\/10.64898\/2026.06.30.735569","relation":{},"subject":[],"published":{"date-parts":[[2026,7,5]]},"subtype":"preprint"}}