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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., Competing Interests: Competing interests: 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., (© 2025. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Abstract: Relapse after CD19-directed chimeric antigen receptor (CAR)-modified T cells remains a substantial challenge. Short CAR T-cell persistence contributes to relapse risk, necessitating novel approaches to prolong durability. CAR T-cell reinfusion (CARTr) represents a potential strategy to reduce the risk of or treat relapsed disease after initial CAR T-cell infusion (CARTi). We conducted a retrospective review of reinfusion of murine (CTL019) or humanized (huCART19) anti-CD19/4-1BB CAR T cells across 3 clinical trials or commercial tisagenlecleucel for relapse prevention (peripheral B-cell recovery [BCR] or marrow hematogones ≤6 months after CARTi), minimal residual disease (MRD) or relapse, or nonresponse to CARTi. The primary endpoint was complete response (CR) at day 28 after CARTr, defined as complete remission with B-cell aplasia. Of 262 primary treatments, 81 were followed by ≥1 reinfusion (investigational CTL019, n = 44; huCART19, n = 26; tisagenlecleucel, n = 11), representing 79 patients. Of 63 reinfusions for relapse prevention, 52% achieved CR (BCR, 15/40 [38%]; hematogones, 18/23 [78%]). Lymphodepletion was associated with response to CARTr for BCR (odds ratio [OR], 33.57; P = .015) but not hematogones (OR, 0.30; P = .291). The cumulative incidence of relapse was 29% at 24 months for CR vs 61% for nonresponse to CARTr (P = .259). For MRD/relapse, CR rate to CARTr was 50% (5/10), but 0/8 for nonresponse to CARTi. Toxicity was generally mild, with the only grade ≥3 cytokine release syndrome (n = 6) or neurotoxicity (n = 1) observed in MRD/relapse treatment. Reinfusion of CTL019/tisagenlecleucel or huCART19 is safe, may reduce relapse risk in a subset of patients, and can reinduce remission in CD19+ relapse., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The use of lentiviral vectors in cell and gene therapy is steadily increasing, both in commercial and investigational therapies. Although existing data increasingly support the usefulness and safety of clinical-grade lentiviral vectors used in cell manufacturing, comprehensive studies specifically addressing their long-term stability are currently lacking. This is significant considering the high cost of producing and testing GMP-grade vectors, the limited number of production facilities, and lengthy queue for production slots. Therefore, an extended shelf life is a critical attribute to justify the investment in large vector lots for investigational cell therapies. This study offers a thorough examination of essential stability attributes, including vector titer, transduction efficiency, and potency for a series of clinical-grade vector lots, each assessed at a minimum of 36 months following their date of manufacture. The 13 vector lots included in this study were used for cell product manufacturing in 16 different clinical trials, and at the time of the analysis had a maximum storage time at -80°C of up to 8 years. The results emphasize the long-term durability and efficacy of GMP-grade lentiviral vectors for use in ex vivo cell therapy manufacturing., Competing Interests: C.H.J is an inventor on patents and/or patent applications licensed to Novartis Institutes of Biomedical Research and receives license revenue from such licenses. C.H.J. is a scientific cofounder of Capstan Therapeutics, Bluewhale Bio, and Tceleron; is a consultant to Kite Pharma; and is a member of the Scientific Advisory Boards of AC Immune, Alaunos, BluesphereBio, Cabaletta, Carisma, Cartography Biosciences, Cellares, Celldex, Decheng, Poseida, Replay Bio, Verismo, and WIRB-Copernicus Group. A.C. is a scientific cofounder of Tceleron, and is a consultant to Kite Pharma and Bluewhale Bio. J.A.F. has received grants and personal fees from Cartography Biosciences, grants from Tmunity Therapeutics, and personal fees from Retro Bio and Shennon Bio outside the submitted work. In addition, J.A.F. holds patents related to CAR T cells for cancer that are licensed and associated with royalties., (© 2024 The Author(s).)

We conducted a phase I clinical trial of anti-BCMA chimeric antigen receptor T cells (CART-BCMA) with or without anti-CD19 CAR T cells (huCART19) in multiple myeloma (MM) patients responding to third- or later-line therapy (phase A, N = 10) or high-risk patients responding to first-line therapy (phase B, N = 20), followed by early lenalidomide or pomalidomide maintenance. We observed no high-grade cytokine release syndrome (CRS) and only one instance of low-grade neurologic toxicity. Among 15 subjects with measurable disease, 10 exhibited partial response (PR) or better; among 26 subjects responding to prior therapy, 9 improved their response category and 4 converted to minimal residual disease (MRD)-negative complete response/stringent complete response. Early maintenance therapy was safe, feasible, and coincided in some patients with CAR T-cell reexpansion and late-onset, durable clinical response. Outcomes with CART-BCMA + huCART19 were similar to CART-BCMA alone. Collectively, our results demonstrate favorable safety, pharmacokinetics, and antimyeloma activity of dual-target CAR T-cell therapy in early lines of MM treatment., Significance: CAR T cells in early lines of MM therapy could be safer and more effective than in the advanced setting, where prior studies have focused. We evaluated the safety, pharmacokinetics, and efficacy of CAR T cells in patients with low disease burden, responding to current therapy, combined with standard maintenance therapy. This article is highlighted in the In This Issue feature, p. 101., (©2022 The Authors; Published by the American Association for Cancer Research.)

Chimeric antigen receptor (CAR) T cells have demonstrated promising efficacy, particularly in hematologic malignancies. One challenge regarding CAR T cells in solid tumors is the immunosuppressive tumor microenvironment (TME), characterized by high levels of multiple inhibitory factors, including transforming growth factor (TGF)-β. We report results from an in-human phase 1 trial of castration-resistant, prostate cancer-directed CAR T cells armored with a dominant-negative TGF-β receptor (NCT03089203). Primary endpoints were safety and feasibility, while secondary objectives included assessment of CAR T cell distribution, bioactivity and disease response. All prespecified endpoints were met. Eighteen patients enrolled, and 13 subjects received therapy across four dose levels. Five of the 13 patients developed grade ≥2 cytokine release syndrome (CRS), including one patient who experienced a marked clonal CAR T cell expansion, >98% reduction in prostate-specific antigen (PSA) and death following grade 4 CRS with concurrent sepsis. Acute increases in inflammatory cytokines correlated with manageable high-grade CRS events. Three additional patients achieved a PSA reduction of ≥30%, with CAR T cell failure accompanied by upregulation of multiple TME-localized inhibitory molecules following adoptive cell transfer. CAR T cell kinetics revealed expansion in blood and tumor trafficking. Thus, clinical application of TGF-β-resistant CAR T cells is feasible and generally safe. Future studies should use superior multipronged approaches against the TME to improve outcomes., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα ( TRAC ) and TCRβ ( TRBC ), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1 ), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Replication-competent retrovirus/lentivirus (RCR/L) and insertional oncogenesis are potential safety risks with integrating viruses in gene-modified cell therapies. As such, the Food and Drug Administration guidances outline RCR/L-monitoring methods throughout the entire gene therapy treatment cycle. We present data for 17 vector lots, 375 manufactured T cell products, and 308 patients post-infusion across both HIV and oncology indications, showing no evidence of RCR/L. Given our data, a Poisson probability model estimates that a single patient, or a group of patients, would need to be followed for at least 52.8 years to observe one positive RCR/L event, highlighting the unlikelihood of RCR/L development. Additionally, we estimate the median time for lentivirus-modified T cell products to fall below the 1% vector sequence threshold in peripheral or whole blood that would trigger vector integration site analysis. These estimated times are 1.4 months in hematologic malignancies, 0.66 month in solid tumors, and 0.92 month in HIV. Based on these considerable safety data in HIV and oncology and recent Biologics License Applications filed for lentiviral-modified T cell products for hematologic malignancies, this may be an opportune time to re-evaluate the current guidelines for T cell gene therapy product testing and long-term patient monitoring., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Chimeric antigen receptor (CAR)-modified T cells with anti-CD19 specificity are a highly effective novel immune therapy for relapsed/refractory acute lymphoblastic leukemia. Cytokine release syndrome (CRS) is the most significant and life-threatening toxicity. To improve understanding of CRS, we measured cytokines and clinical biomarkers in 51 CTL019-treated patients. Peak levels of 24 cytokines, including IFNγ, IL6, sgp130, and sIL6R, in the first month after infusion were highly associated with severe CRS. Using regression modeling, we could accurately predict which patients would develop severe CRS with a signature composed of three cytokines. Results were validated in an independent cohort. Changes in serum biochemical markers, including C-reactive protein and ferritin, were associated with CRS but failed to predict development of severe CRS. These comprehensive profiling data provide novel insights into CRS biology and, importantly, represent the first data that can accurately predict which patients have a high probability of becoming critically ill., Significance: CRS is the most common severe toxicity seen after CAR T-cell treatment. We developed models that can accurately predict which patients are likely to develop severe CRS before they become critically ill, which improves understanding of CRS biology and may guide future cytokine-directed therapy. Cancer Discov; 6(6); 664-79. ©2016 AACR.See related commentary by Rouce and Heslop, p. 579This article is highlighted in the In This Issue feature, p. 561., (©2016 American Association for Cancer Research.)

Background: Relapsed acute lymphoblastic leukemia (ALL) is difficult to treat despite the availability of aggressive therapies. Chimeric antigen receptor-modified T cells targeting CD19 may overcome many limitations of conventional therapies and induce remission in patients with refractory disease., Methods: We infused autologous T cells transduced with a CD19-directed chimeric antigen receptor (CTL019) lentiviral vector in patients with relapsed or refractory ALL at doses of 0.76×10(6) to 20.6×10(6) CTL019 cells per kilogram of body weight. Patients were monitored for a response, toxic effects, and the expansion and persistence of circulating CTL019 T cells., Results: A total of 30 children and adults received CTL019. Complete remission was achieved in 27 patients (90%), including 2 patients with blinatumomab-refractory disease and 15 who had undergone stem-cell transplantation. CTL019 cells proliferated in vivo and were detectable in the blood, bone marrow, and cerebrospinal fluid of patients who had a response. Sustained remission was achieved with a 6-month event-free survival rate of 67% (95% confidence interval [CI], 51 to 88) and an overall survival rate of 78% (95% CI, 65 to 95). At 6 months, the probability that a patient would have persistence of CTL019 was 68% (95% CI, 50 to 92) and the probability that a patient would have relapse-free B-cell aplasia was 73% (95% CI, 57 to 94). All the patients had the cytokine-release syndrome. Severe cytokine-release syndrome, which developed in 27% of the patients, was associated with a higher disease burden before infusion and was effectively treated with the anti-interleukin-6 receptor antibody tocilizumab., Conclusions: Chimeric antigen receptor-modified T-cell therapy against CD19 was effective in treating relapsed and refractory ALL. CTL019 was associated with a high remission rate, even among patients for whom stem-cell transplantation had failed, and durable remissions up to 24 months were observed. (Funded by Novartis and others; CART19 ClinicalTrials.gov numbers, NCT01626495 and NCT01029366.).