Aug 2026· Science Translational Medicine· Vol 18 863, pp.
eaei0875
· 0 citations· 53 references
Medicine
TL;DR
This first-in-human study demonstrated the feasibility of an "off-the-shelf" base-edited CAR T cell approach and informs future multiantigen strategies against AML.
Abstract
Chimeric antigen receptor (CAR) T cell therapy for acute myeloid leukemia (AML) is constrained by antigen heterogeneity and shared expression with healthy compartments, and there are often challenges in obtaining autologous T cells from heavily pretreated patients. To address these challenges, we developed universal donor-derived, base-edited, anti-CD33 CAR T cells (BE-CAR33) that used precise multiplexed cytidine deamination to simultaneously disrupt the TRAC, CD52, and CD7 loci to prevent graft-versus-host disease and evade immunotherapy effects. An open-label, nonrandomized, single-center phase 1 study (ISRCTN14430213) evaluated the safety, feasibility, and activity of BE-CAR33 cell therapy ahead of allogeneic stem cell transplantation (allo-SCT) for patients with AML. Eligible participants were aged less than 16 years with relapsed/refractory AML. Five patients were screened, and three were enrolled; one additional adult received BE-CAR33 through compassionate access. Participants received fludarabine, cyclophosphamide, and alemtuzumab followed by 1.2 to 1.8 × 106 BE-CAR33 cells per kilogram. Treatment-emergent adverse events included cytokine release syndrome (grade ≤2), neurotoxicity (grade 3), cytopenias (grade 4), and transient rashes. Two patients demonstrated reduced minimal residual disease and proceeded to allo-SCT. Serial flow cytometry, chimerism quantification, and vector copy number analyses tracked BE-CAR33 T cells until elimination during transplant. Differentially expressed genes included editing signatures and switched from manufacturing-related toward postexpansion effector and exhaustion profiles. Although primary end points were not met, this first-in-human study demonstrated the feasibility of an "off-the-shelf" base-edited CAR T cell approach and informs future multiantigen strategies against AML.
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis. Current Immunotherapies, including chimeric antigen receptor (CAR) T cell strategies, are limited by the scarcity of antigens that are present on AML cells but absent from hematopoietic stem and progenitor cells (HSPCs). To identify a potential therapeutic target, we analyzed two publicly available single-cell RNA sequencing datasets and found that CD4 is frequently expressed on AML blasts but absent from HSPCs, supporting its potential as a novel CAR target. We developed a CD4-targeted CAR using CD3+CD4⁻CD8⁻ double-negative T cells (CAR4-DNTs). DNTs offer a unique therapeutic platform: they naturally lack CD4, preventing fratricide, and elicit endogenous anti-leukemic activity. Their feasibility and safety as an allogeneic cell therapy platform was demonstrated in a Phase I trial. This work aims to establish CAR4-DNTs as a next-generation, off-the-shelf CAR-T cell therapy for AML.
Donor-derived DNTs were engineered to express a CD4-CAR. Cytotoxicity, persistence, and cytokine secretion were evaluated against AML cell lines, CD4+ and CD4-knockout AML variants, and primary AML samples. NSG-SGM3 mice engrafted with luciferase+ MV4-11 cells received CAR4-DNTs, untransduced (UT) DNTs, or PBS. Disease progression was monitored by bioluminescence imaging and survival analysis.
CAR4-DNTs were successfully manufactured without fratricide. They exhibited significantly enhanced and more durable cytotoxicity than UT-DNTs against AML cell lines and patient samples. Importantly, CAR4-DNTs retained endogenous cytotoxicity towards CD4-negative AML cells. In vivo, CAR4-DNT achieved durable leukemia clearance and prolonged survival, whereas PBS-treated mice rapidly progressed and UT-DNT recipients showed transient disease control before relapse.
These findings establish CAR4-DNTs as a promising immunotherapy candidate for AML, supporting their further preclinical and translational development.
Distinguished Doctoral Recruitment Scholarship, The C17 Council, Alberta Cancer Foundation, Alberta Children’s Hospital Research Institute
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Shanshan Wang, Michele Nawata, Juan Arteaga et al.· Journal of Immunology· 0 citations
PURPOSE Treatment options for clear cell renal cell carcinoma (ccRCC) refractory to immune checkpoint inhibitors (ICIs) and targeted therapy are needed. ALLO-316 is an allogeneic chimeric antigen receptor (CAR) T-cell product that targets CD70-expressing ccRCC and resists immune rejection by targeting and eliminating patients' CD70+ alloreactive T cells. METHODS In the phase Ia/b TRAVERSE trial (ClinicalTrials.gov identifier: NCT04696731), patients with advanced ccRCC resistant to ICIs and vascular endothelial growth factor receptor–targeted therapy received lymphodepletion and ALLO-316 following a modified 3 + 3 design. Phase Ia evaluated ALLO-316 dose and fludarabine/cyclophosphamide (FC)–based lymphodepletion with/without the anti-CD52 antibody, ALLO-647. Additional patients were enrolled in phase Ib to confirm the expansion regimen identified in phase Ia. Primary end points were dose-limiting toxicities (DLTs) and adverse events (AEs). RESULTS Fifty-one patients were enrolled (phase Ib, n = 23). Patients received a median of four prior lines of therapy. The median follow-up was 28.8 months. DLTs occurred in two patients who received ALLO-647 (grade 3 autoimmune hepatitis; grade 5 cardiogenic shock). Grade ≥3 cytokine release syndrome, immune effector cell–associated neurotoxicity syndrome, and immune effector cell–associated hemophagocytic lymphohistiocytosis-like syndrome occurred in 2.0%, 0%, and 6.0% of patients, respectively. Most grade ≥3 AEs were hematologic (neutropenia 62.0%; white blood cell count decreased 54.0%; anemia 36.0%). In phase Ib, patients received FC and 80 × 106 CAR T cells. The objective response rate was 17.4% overall, 25.0% in phase Ib patients and 31.3% in phase Ib patients with CD70 tumor proportion score ≥50%. CONCLUSION ALLO-316 had manageable safety and encouraging antitumor activity in CD70-positive ccRCC. The TRAVERSE trial demonstrates proof of concept for the role of allogeneic CAR T-cell therapy in solid tumors.
S. Srour, J. Chahoud, A. Drakaki et al.· Journal of Clinical Oncology· 0 citations
Disease recurrence is the main cause of treatment failure after CD19-directed CAR T cells, often due to CD19 antigen loss, stability and/or coverage. To overcome single-antigen escape, we evaluated a trispecific CAR targeting CD19, CD20, and CD22 with OX40 co-stimulatory domain. Preclinical studies demonstrated potent, antigen-specific cytotoxicity in in vitro and in vivo lymphoma models. We then conducted a first-in-human phase I trial in patients with relapsed/refractory B-cell malignancies. Sixteen patients received infusions at a median vein-to-vein time of 7 days, at doses of 0.5-2×10⁶ cells/kg. No severe cytokine release syndrome nor neurotoxicity occurred. Overall response rate was 50%, including complete responses in 83% of lymphoma patients. One-year overall survival rate was 61%, with durable remissions observed in lymphoma. CAR T expansion did not correlate with dose or response. T-cell exhaustion in apheresis cells correlated with progressive disease. Trispecific CAR T cells are safe and potentially active in lymphoma.
S. Vasu, N. Denlinger, No-Joon Song et al.· Blood Cancer Discovery· 0 citations
BACKGROUND
B cell-targeting chimeric antigen receptor (CAR) T cell therapy has shown efficacy in autoimmune diseases but is limited by toxicity, complex manufacturing, and high cost. Umbilical cord blood (UCB)-derived CAR-natural killer (CAR-NK) cells offer an alternative "off-the-shelf" platform with a potentially superior safety profile. We developed a UCB-derived CD19-targeting CAR-NK product incorporating the 4-1BB costimulatory domain (CD19-BBz) and evaluated its safety and efficacy in refractory systemic lupus erythematosus (SLE). This study was registered at ClinicalTrials.gov (ClinicalTrials.gov: NCT06421701).
METHODS
In this phase 1, open-label, dose-escalation study, five patients with refractory SLE received lymphodepletion chemotherapy followed by infusion of allogeneic CD19-BBz CAR-NK cells. Dosing followed a step-up regimen, with the highest total dose being 1.35 × 109 cells-2.2- to 3.3-fold lower than the highest doses previously reported for CAR-NK therapy in SLE.
FINDINGS
Treatment was exceptionally well tolerated. No grade ≥2 cytokine release syndrome and no neurotoxicity or graft-versus-host disease occurred. All patients achieved profound B cell depletion, with nadir circulating B cell levels ranging from 0.16 to 1.44 cells/μL. All patients achieved an SLE Responder Index-4 response by month 1. The lupus low disease activity state was attained in all patients by month 9, and 80% (4/5) met the definition of remission in SLE by the last follow-up (median, 12 months). Reconstituted B cells displayed a sustained naive-dominant repertoire.
CONCLUSIONS
Low-dose UCB-derived CD19-BBz CAR-NK cell therapy demonstrated an excellent safety profile and induced robust, durable clinical responses in refractory SLE.
FUNDING
This study was supported by the Noncommunicable Chronic Diseases-National Science and Technology Major Project (2023ZD0501300).
Zhiru Zeng, W. Lei, Wenhai Deng et al.· i Medicina· 1 citation
CAR-T therapy targeting B-cell antigens is effective for relapsed hematologic malignancies, but antigen escape remains a challenge. We previously showed dual CD19/CD20-4-1BB CAR-T therapy was highly effective (Nat Med, 2020). We further developed trispecific CAR-T cells (CD19/CD20/CD22) incorporating ICOS signaling (Sci Transl Med, 2021). Despite promising preclinical activity, trispecific-ICOS CAR-T cells failed to expand and showed no efficacy in a Phase I trial (NCT05094206; ASH 2024). Here, we investigated mechanisms underlying this failed response.
Based on our IRB approved protocol and using patient-derived PBMCs and leftover samples from treated patients, we generated tested bispecific-4-1BB, bispecific-ICOS and trispecific-ICOS CAR-T cells.
NSG mice receiving bispecific- or trispecific-ICOS CAR-T cells had worse survival than those with 4-1BB CAR-T cells. In vivo, ICOS CAR-T cells failed to expand and showed an exhaustion phenotype. When co-culture with Raji cells, ICOS CAR-T cells significantly enhanced surface-FMC63 loss compared to 4-1BB counterparts. Surface-FMC63 expression was mostly recovered at 40 hours on 4-1BB CAR-T cells, but not on ICOS CAR-T cells. Since trogocytosis can mediate CAR internalization and antigen loss on tumor, we examined CD19 transfer. ICOS CAR-T cells displayed higher and more sustained CD19 uptake and fratricide reflected by apoptosis than 4-1BB cells. Consistently, when co-culture with patient autologous B cells, ICOS CAR-T cells again showed greater CAR loss and apoptosis. Extended co-culture mimicking chronic antigen stimulation revealed expansion of 4-1BB but not ICOS CAR-T cells, the latter showing reduced proliferation (Ki67) and elevated exhaustion markers.
In conclusion, the ICOS domain promotes trogocytosis and fratricide, explaining the poor persistence and function of ICOS CAR-T cells observed in our trial. Thus, our findings raise significant caution for using CAR constructs that rely on ICOS for CAR activation.
n/a
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Yongxia Wu, Allison Pugel, Katie A. Palen et al.· Journal of Immunology· 0 citations
BACKGROUND
Chimeric antigen receptor (CAR) T-cell therapies have transformed the treatment of B-cell non-Hodgkin lymphoma, but centralised manufacturing-with its complex logistics and long vein-to-vein times-drives up costs and restricts access. We aimed to evaluate the safety of GLPG5101, a fresh, CD19 CAR T-cell product manufactured through a decentralised process, and determine the recommended phase 2 dose.
METHODS
This phase 1 dose-escalation part of the ATALANTA-1 phase 1/2, single-arm study, was executed in five hospitals in the Netherlands and Belgium. Patients aged 18 years or older, with histologically confirmed diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma (MZL), or mantle cell lymphoma (MCL) after two or more lines of therapy, measurable disease according to the Lugano classification, Eastern Cooperative Oncology Group Performance Status 0-2, and adequate organ function were enrolled. Patients were treated with a single intravenous infusion at one of three dose levels of GLPG5101 (dose level 1 [35-50 × 106 viable CAR+ T-cells], dose level 2 [85-110 × 106 viable CAR+ T-cells], and dose level 3 [200-250 × 106 viable CAR+ T-cells]). The phase 1 part of the study applied a Bayesian Optimal Interval design and the primary endpoints were safety (incidence of adverse events and serious adverse events until end of treatment [week 14], and dose-limiting toxicities [DLTs] until day 28) in patients who received fresh GLPG5101 at any dose (excluding recipients of non-conforming product [not meeting prespecified release criteria other than dose]) and determination of the recommended phase 2 dose. The study was registered with ClinicalTrials.gov (NCT06561425) and is closed for recruitment.
FINDINGS
From March 15, 2022, to Sept 10, 2024, 27 patients were screened for eligibility, 24 of whom were enrolled, underwent leukapheresis and lymphodepleting chemotherapy and received GLPG5101. Data cutoff was June 1, 2025. In the intention-to-treat population (n=24), the median age was 66·5 years (IQR 59·0-71·5), 15 (63%) patients were male, nine (38%) were female, and 21 (88%) were White. One of the 24 patients received a non-conforming product and was excluded from the safety analysis population. Median follow-up was 24·0 months (IQR 20·7-24·9). Five DLTs were reported: grade 3 thrombocytopenia (n=1, dose level 1), death from intra-abdominal haemorrhage (n=1, dose level 2), and grade 4 prolonged neutropenia not resolving to grade 2 or lower within 28 days (n=1 at dose level 1 and n=2 at dose level 2). All 23 patients in the safety analysis population had grade 3 or higher treatment-emergent adverse events; the most common were neutropenia (22 [96%]), leukopenia (nine [39%]), lymphopenia (seven [30%]), anaemia (six [26%]), and thrombocytopenia (five [22%]). Treatment-related deaths occurred in four patients: one during the 14-week treatment period due to intra-abdominal haemorrhage, and three after the treatment period due to Escherichia coli sepsis (n=1), immune-effector cell-associated haemophagocytic lymphohistiocytic syndrome (n=1), and COVID-19 (n=1). The safety review committee selected 110 × 106 (range 50-110 × 106) viable CAR T-cells as the recommended phase 2 dose.
INTERPRETATION
The results of this study showed the feasibility of rapid multicentre decentralised manufacturing and delivery of fresh CAR T-cell therapy in heavily pretreated B-cell non-Hodgkin lymphoma. The phase 2 part of ATALANTA-1 will provide further information on clinical activity and safety.
FUNDING
CellPoint, Lakefront Biotherapeutics.
M. Kersten, M. Kuipers, P. Mutsaers et al.· The Lancet Haematology· 0 citations
A new method for surgically removing training examples from a model reveals that as datasets grow, the link between what a model learns and what it produces dissolves.
