Abstract
Long-term risks of gene therapy are not fully understood. In this study, we evaluated safety outcomes in 783 patients over more than 2,200 total patient-years of observation from 38 T cell therapy trials. The trials employed integrating gammaretroviral or lentiviral vectors to deliver engineered receptors to target HIV-1 infection or cancer. Eighteen patients (2.3%) developed secondary malignancies after treatment, with a median onset of 1.94 years (range: 51 d to 14 years). Where possible, incident tumor samples were analyzed for vector copy number, revealing no evidence of high-level marking or other indications of insertional mutagenesis. One T cell lymphoma was detected, but malignant T cells were not marked by vector integration. Analysis of vector integration sites in 176 patients revealed no pathological insertions linked to secondary malignancies, although, in some cases, integration in or near specific genes, including tumor suppressor genes, was associated with modest clonal expansion and sustained T cell persistence. These findings highlight the safety of engineered T cell therapies.
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Data availability
All study data are available within the main paper, extended data and supplementary materials. The integration site sequencing data have been deposited in the Sequence Read Archive (SRA) hosted by the National Center for Biotechnology Information. The accession number for the SRA dataset is PRJNA1110224. Sample metadata are further summarized in Supplementary Data Table 1. Publicly available datasets used in this study include dbSNP, COSMIC, gnomAD and the 1000 Genomes Project, which were accessed through their respective repositories. For inquiries regarding additional data or materials, please contact the Penn Center for Innovation at pciinfo@pci.upenn.edu. Requests will be addressed within 2 weeks to ensure compliance with intellectual property and confidentiality agreements. Data and materials will be shared after the completion of a material transfer agreement. For further assistance, please contact the corresponding authors.
Code availability
Code supporting the integration site analyses performed in this study is available at https://github.com/helixscript/CART_safety_paper_2024.
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Acknowledgements
We extend our sincere gratitude to the patients and their families for participating in the clinical trials that form the foundation of this study, supported by the University of Pennsylvania. We especially thank L. Zimmerman for her invaluable assistance with figures. Additionally, we acknowledge the Hospital of the University of Pennsylvania Apheresis Unit for its role in collecting peripheral blood T cells. We extend our appreciation to the data safety monitoring and institutional review boards, the Institutional Biosafety Committee and various core groups at the University of Pennsylvania Center for Cellular Immunotherapies. Special thanks go to the Clinical Cell and Vaccine Production Facility, with particular recognition for T. Colligon and the Manufacturing, Quality Control and Quality Assurance Teams. Additionally, we acknowledge the contributions of the Clinical Trials Unit, the Clinical Operations Group, the Translational Scientific Operations Group, the Translational and Correlative Sciences Laboratory, the Human Genome Editing Laboratory and the Product Development Laboratory to the conduct of the clinical studies. We also thank S. Emanuel and the entire team at the University of Pennsylvania Office of Clinical Research. Finally, we are grateful to all members of the Fraietta and Bushman laboratories, including D. Jarocha, E. Thai, C. Nobles, D. Kardeniz, F. Male, H. Raymond, H. Adhikari, L. Kessler and P. Hokama, for their support and insights. Thanks also to E. Marcuson and L. Encarnacion for help with clinical data collection. Á.R.-F. receives funding from the AIDS Malignancy Consortium and the Alfonso Martín Escudero Foundation. A.L.G. receives support from a Leukemia & Lymphoma Society Scholar in Clinical Research Award. This study additionally received funding from National Institutes of Health grants R01 CA241762 (to W.-T.H., J.J.M., F.D.B. and J.A.F.) and P01 CA214278 (to W.-T.H., M.R., D.L.S., C.H.J. and J.A.F.) and the Bob Levis Funding Group (to S.J.S., B.L.L., C.H.J. and J.A.F.). Further support was provided by the National Science Foundation Engineering Research Center for Cell Manufacturing Technologies (to B.L.L. and J.A.F.), the Alliance for Cancer Gene Therapy Investigator Award in Cell and Gene Therapy for Cancer (to J.A.F.) and funding from the Parker Institute for Cancer Immunotherapy (to C.H.J.). J.A.F. also receives funding for correlative data science from a Parker Institute for Cancer Immunotherapy Innovation Challenge Award. Additionally, funding was received from U01 AG066100 via the Samuel Waxman Cancer Research Foundation and a Chambers Centurion gift (to C.H.J. and J.A.F.), Abramson Cancer Center Core Grant P30 CA016520 (to W.-T.H. and J.A.F.) and a Prostate Cancer Foundation Tactical Award (to C.H.J. and J.A.F.).
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J.K.J and E.O.H. are co-first authors. J.K.J., E.O.H., A.M., S.A.G., N.V.F., S.L.M., C.H.J., F.D.B. and J.A.F. participated in the design, execution and/or interpretation of the reported experiments or results. J.K.J., E.O.H., A.M., S.A.G., N.V.F., J.L.R., E.V., H.M., W.R., C.C., W.-T.H., Y.L., R.M.L., O.F., J.K., S.C., J.B.-R., A.G., N.A., W.B., J.J.M., A.M.R., J.K.E., S.A.Z., A.G.M., S.R., A.P., E.J.C., C.L., V.E.G., K.A., I.K., Á.R.-F., J.C.M., M.R., S.I.G., S.J.S., A.D.C., A.L.G., P.D.S., J.L.T., D.L.P., B.L.L., D.L.S., A.C., S.M., L.L., M.M.D., G.P., F.H., E.A.S., P.T., A.D., A.H., N.B.H., R.M., D.M.O., J.S., J.L.T., R.A., J.M.J., A.H.K. and R.B.C. participated in the acquisition or analysis of data. J.K.J., E.O.H., F.D.B. and J.A.F. wrote the paper, with all authors contributing to writing and providing feedback. C.H.J., F.D.B. and J.A.F. supervised all aspects of the research.
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E.O.H.: consulting or advisory role with Blueprint Medicines and the American Board of Internal Medicine Subspecialty Board; research funding from Blueprint Medicines and Tmunity Therapeutics. J.J.M.: fees received from IASO Biotherapeutics, Poseida Therapeutics and Kite Pharma unrelated to this work; holds patents related to enhancing immune cell efficacy and predicting chimeric antigen responsiveness issued to Novartis. S.A.G.: support received and advisory capacities served for multiple entities within the pharmaceutical sector, including Novartis and Servier. N.V.F., D.L.P. and F.D.B.: engaged with Sana Biotechnology; N.V.F. also holds consultancy roles with Novartis and Syndax Pharmaceuticals and obtains funding from Kite Pharma. J.L.R.: grants received from Tmunity/Kite outside the submitted work and is a co-founder of Tmunity Therapeutics and BlueWhale Bio with monetary compensation and equity. M.R.: patents related to CD19 CAR-T cells; consultant for NanoString, Bristol Myers Squibb, GlaxoSmithKline, Scaylite, Bayer and AbClon; research funding received from AbClon, NanoString, Oxford NanoImaging, viTToria Biotherapeutics, CURIOX and Beckman Coulter; scientific founder of viTToria Biotherapeutics. S.I.G.: stock ownership interests in Carisma Therapeutics; advisory role with Asher Bio; research funding from Carisma Therapeutics and Novartis; holds patents for chimeric antigen receptor T cells for acute myeloid leukemia. S.J.S.: consultant to various companies, including AstraZeneca, BeiGene, Celgene, Genentech, Genmab, Fate Therapeutics, Roche, Incyte, Juno Therapeutics, Legend Biotech, Loxo Oncology, MorphoSys, Mustang Biotech, Nordic Nanovector, Novartis and Regeneron; research funding received from AbbVie, Adaptive Biotechnologies, Celgene, DTRM, Genentech, Roche, Juno Therapeutics, Merck, Novartis, Incyte, Pharmacyclics and TG Therapeutics; honoraria from Celgene and Novartis; holds patents related to CD19 CAR-T cells and autologous co-stimulated T cells. A.D.C.: consulting with Janssen, GlaxoSmithKline, Bristol Myers Squibb/Celgene, Genentech/Roche, Pfizer, AbbVie, Arcellx and Ichnos. A.L.G.: consulting with Bristol Myers Squibb, Janssen, Novartis, GlaxoSmithKline, AbbVie, Regeneron, Gracell Bio and Legend; research funding from Novartis, Janssen, Tmunity and CRISPR Therapeutics; patents in CAR-T cell therapy with Novartis. P.D.S.: stock ownership interests in Johnson & Johnson, Novartis, Novo Nordisk, Pfizer, Merck and Amgen; consulting or advisory role with Tmunity Therapeutics. D.L.P.: membership on advisory boards for the National Marrow Donor Program, Kite/Gilead, Janssen, Incyte, Sana Biotechnology and Verismo; equity holder in Genentech; honoraria from the American Society for Transplantation and Cellular Therapy; patents and royalties with Novartis, Tmunity and Wiley and Sons Publishing. S.L.M.: clinical trial support and advisory roles with Novartis and Wugen; pending patent with Novartis. B.L.L.: consultancy and advisory positions with Terumo, GlaxoSmithKline and Kite; co-founder and equity holder in Tmunity Therapeutics (acquired by Kite) and Capstan Therapeutics; membership on advisory boards for Avectas, Capstan Therapeutics (chair), Immuneel, Immusoft, In8bio, Ori Biotech, Oxford Biomedica, Thermo Fisher Pharma Services and UTC Therapeutics; Alliance for Cancer Gene Therapy Board of Directors. D.L.S.: founder’s equity and licensed intellectual property to Verismo Therapeutics, Vetigenics and Chimeric Therapeutics. A.C.: co-founder with equity in Tmunity Therapeutics. M.M.D.: intellectual property and patent rights concerning T-cell-based cancer immunotherapy, including royalty payments; research funding from Tmunity Therapeutics; member of the scientific advisory board for Cellares Corporation. F.H.: research funding from Danaher and Kite. E.A.S.: consultant for Janssen and Bristol Myers Squibb; grant funding from Sorrento and AbbVie. A.R.H.: consulting or advisory role with Olympus America and Novocure. N.B.H.: consulting or advisory role with Pfizer, Merck Sharp & Dohme, Calithera Biosciences, Eisai, Exelixis, AVEO and Roche/Genentech; expert testimony with Eli Lilly. D.M.O.: inventor of intellectual property and received royalties related to targeted ErbB therapy in solid cancers and CAR-T cell therapy in solid tumors; inventor on multiple patents licensed by the University of Pennsylvania. J.S.: consulting or advisory role with Seattle Genetics, Bristol Myers Squibb, AstraZeneca, Pharmacyclics and Imbrium; research funding from Celgene, Seattle Genetics, Pharmacyclics, Merck, Bristol Myers Squibb, Incyte and AstraZeneca; research funding from Kite. R.A.: honoraria from Sigma-Tau; expert testimony with Wiggin and Dana. A.H.K.: honoraria from various organizations; consulting or advisory roles with ERYTECH Pharma, Imugene, SynCoreBio, Gritstone Bio, Roche/Genentech, Ipsen, Five Prime Therapeutics, Tyme, Turning Point Therapeutics, Signatera and Syros Pharmaceuticals; speakers’ bureau with Clinical Care Options; research funding from Celgene, Merck, Genentech/Roche, Bristol Myers Squibb, AbGenomics International, Apexigen and Astellas Pharma. R.B.C.: consulting or advisory role with Heat Biologics, Takeda, Alkermes, Kyntherapeutics, Innate Pharma, Cantargia, Genocea Biosciences and AstraZeneca; research funding from Heat Biologics, Merck, Celldex, Innate Pharma, Kyntherapeutics, Xencor and Genocea Biosciences. C.H.J.: royalties paid from Novartis and Kite to the University of Pennsylvania; scientific co-founder and equity holder in Capstan Therapeutics, Dispatch Biotherapeutics and BlueWhale Bio; board membership with AC Immune; scientific advisory roles with various companies, including BlueSphereBio, Cabaletta, Carisma, Cartography, Cellares, Cellcarta, Celldex, Danaher, Decheng, ImmuneSensor, Kite, Poseida, Verismo, Viracta and WIRB-Copernicus group. J.A.F.: patents and intellectual property in T cell-based cancer immunotherapy with royalties; funding from Tmunity Therapeutics and Danaher; consultancy with Retro Biosciences; scientific advisory board memberships with Cartography Bio, Shennon Biotechnologies, CellFe Biotech, OverT Bio and Tceleron Therapeutics. All other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Vector copy number analysis in transduced T cell samples.
Dot plot illustrating the vector copy number (VCN) values for all lentiviral CAR- T cell products (pre-infusion) and their corresponding longitudinal time points post-product infusion into patients, where data are available.
Supplementary information
Supplementary Tables 1 and 4
Supplementary Data Table 1 summarizes metadata, integration site metrics and Sequence Read Archive (SRA) accession numbers for T cell therapy subjects across clinical trials, including sample and patient identifiers, sample types and inferred cell populations. Supplementary Data Table 4 lists linker and PCR primer sequences used for integration site analyses, including linker designations and primer sequences for the first and second rounds of PCR.
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Jadlowsky, J.K., Hexner, E.O., Marshall, A. et al. Long-term safety of lentiviral or gammaretroviral gene-modified T cell therapies. Nat Med 31, 1134–1144 (2025). https://doi.org/10.1038/s41591-024-03478-6
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DOI: https://doi.org/10.1038/s41591-024-03478-6
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