Next-generation CAR-T therapy for acute myeloid leukemia: bridging innovation with clinical translation.

Introduction

Acute myeloid leukemia (AML) is a genetically and phenotypically heterogeneous malignancy characterized by clonal proliferation of immature myeloid cells and frequent relapse [ 1 ]. Despite advances in chemotherapy, targeted agents, and allogeneic hematopoietic stem cell transplantation (allo-HSCT), durable remissions remain uncommon in high-risk patients, underscoring the need for alternative strategies [ 2 – 4 ].

Chimeric antigen receptor (CAR) T-cell therapy has transformed outcomes in B-cell malignancies, but its application to AML is hindered by challenges intrinsic to myeloid disease. Most candidate antigens, including CD33 and CD123, are expressed on both leukemic cells and normal hematopoietic progenitors, restricting the therapeutic window due to inevitable myelotoxicity [ 5 ]. Antigen expression is heterogeneous and dynamically regulated, facilitating immune escape [ 6 ]. The bone marrow microenvironment—rich in regulatory T cells, myeloid-derived suppressor cells, and inhibitory cytokines—further accelerates T-cell exhaustion [ 7 ]. Clinically, high leukemic burden and rapid disease progression limit the window for autologous manufacturing, while blast contamination and poor T-cell quality reduce manufacturing success [ 8 ]. The timing of infusion, whether during active disease or remission, involves trade-offs between efficacy, toxicity, and persistence.

This review examines current and emerging AML antigens in relation to their compatibility with next-generation CAR-T platforms, and presents a translational framework that integrates target selection, engineered design, and clinical sequencing to address the biological, manufacturing, and application barriers unique to AML.

Targets for CAR-T therapy in AML

The clinical application of CAR-T therapy in AML remains limited by the absence of antigens that can reliably differentiate leukemic cells from normal hematopoietic progenitors. An optimal CAR-T target is expected to be strongly expressed on AML blasts and leukemic stem cells (LSCs), with negligible expression on normal hematopoietic stem and progenitor cells (HSPCs) to minimize on-target, off-leukemia toxicity [ 8 ]. However, many candidate antigens also appear on healthy myeloid cells, leading to prolonged marrow suppression. The extensive molecular and phenotypic heterogeneity of AML further complicates the discovery of a universal target [ 9 ].

More recent studies have focused on targets such as CD123, CD33, and CLEC12A, along with emerging candidates like CD70, FLT3, NKG2DL, and TIM-3 (Table 1 ). Tumor-specific glycoforms have also gained interest as a strategy to improve selectivity. While these newer targets demonstrate potent anti-leukemic activity in vitro and in animal models, only a subset have progressed into clinical evaluation, and most remain in the preclinical stage (Tables 2 and 3 ). This highlights the persistent gap between target discovery and clinical translation.

Table 1 Characteristics of various AML target molecules Target antigens Molecule type Function Research phase In AML On normal cells On HSCs On LSCs On AML blasts Challenges Optimization Strategies References CD123 Type Ⅰ cytokine receptor IL-3 receptor Phase Ⅰ/Ⅱ clinical trial 40–93% of cases Myeloid cells, myeloid progenitors, DC, epithelial cells and basophils + + + Hematopoietic toxicity, Antigen escape Dual-target CAR-T to mitigate antigen escape, Switchable CAR-T to minimize hematopoietic toxicity, HMA (azacitidine/decitabine) to enhance CD123 expression [ 10 – 16 ] CD33 SIGLEC family protein/Glycoprotein Transmembrane receptor Phase Ⅰ/Ⅱ clinical trial 85–90% of cases Myeloid cells, myeloid progenitors and Kupffer cells + + + Myelosuppression, Limited CAR-T persistence Dual-target CAR-T to minimize myelosuppression, Inducible CAR-T to enhance safety [ 17 – 22 ] CLEC12A (CLL-1) Glycoprotein Transmembrane receptor Phase Ⅰ/Ⅱ clinical trial 92% of cases Myeloid cells, myeloid progenitors, and epithelial cells - + + Myelosuppression, Low antigen density Dual-target CAR-T to enhance specificity, HSCT following CAR-T to restore hematopoiesis [ 23 – 30 ] CD38 Glycoprotein Cyclic ADP ribose hydroxylase Phase Ⅰ/Ⅱ clinical trial 91.7% of cases T cells, B cells, NK cells and plasma cells + - + Low antigen density, CAR-T fratricide CRISPR knockout of CD38 in T cells to prevent fratricide, ATRA to enhance CD38 expression [ 31 – 36 ] CD44v6 Glycoprotein Transmembrane receptor Phase Ⅰ/Ⅱ clinical trial 60% of cases Activated T cells, monocytes and Keratinocytes - - + Hematopoietic toxicity, Antigen escape Localized CAR-T delivery to reduce off-target effects, CRISPR-modified CAR-T to enhance antigen specificity [ 37 – 39 ] FLT3 Type Ⅲ cytokine receptor Tyrosine kinase receptor Phase Ⅰ/Ⅱ clinical trial 93% of cases DC and NK cells + + + Low antigen density, Antigen escape FLT3 inhibitors to improve CAR-T cytotoxicity, Safety switch CAR-T to enhance controllability [ 40 – 45 ] SIGLEC-6 SIGLEC family protein/Glycoprotein Inhibitory receptor with ITIM motifs; potential role in immune regulation Phase Ⅰ/Ⅱ clinical trial / Placenta, mast cells, memory B cells, basophils - + + Low antigen density, Immune evasion Checkpoint blockade to improve CAR-T persistence, Dual-target CAR-T to enhance specificity [ 46 – 53 ] CD7 Ig superfamily/Glycoprotein B and T cell lymphoid development, transmembrane protein Phase Ⅰ/Ⅱ clinical trial 30% of cases Activated T cells, NK cells, lymphoid and myeloid progenitors - + + CAR-T fratricide CRISPR knockout of CD7 in T cells to prevent fratricide [ 54 – 63 ] CD70 TNF family/Glycoprotein Transmembrane receptor Phase Ⅰ clinical trial / T cells and B cells - + + Low antigen density, CAR-T fratricide Nanobody-based CAR-T to improve antigen recognition, CRISPR knockout of CD70 in T cells to prevent fratricide [ 64 – 69 ] NKG2DL MICA, MICB and ULBP1-6 Activating immune cells by binding to NKG2D Phase Ⅰ clinical trial 67–100% of AML blasts / - - + Low antigen density, Immune evasion HDAC inhibitors to enhance antigen expression, MMP inhibitors to prevent ligand shedding [ 70 – 78 ] TIM3 Ig superfamily/Glycoprotein Transmembrane protein Phase Ⅰ clinical trial / T cells, NK cells and myeloid cells - + + Immune evasion, Low antigen density PD-1/PD-L1 blockade to improve CAR-T persistence, iPSC-derived CAR-NK cells to enhance cytotoxicity [ 79 – 88 ] LeY Glycosphingolipid Blood group antigen Phase Ⅰ clinical trial 46% of cases Intestinal epithelial cells + + + Low antigen density, Antigen escape ATRA to enhance antigen expression, Dual-target CAR-T to minimize resistance [ 89 – 92 ] ADGRE2 G protein-coupled receptor, adhesion family Mediates cell adhesion and immune regulation Phase Ⅰ clinical trial / Monocytes and macrophages - + + Low antigen density, Antigen escape ATRA to enhance antigen expression, Dual-target CAR-T to minimize resistance [ 93 – 97 ] CD117 Type Ⅲ cytokine receptor Tyrosine kinase receptor, transmembrane protein Preclinical / Myeloid progenitors, erythroid progenitors, mast cells and epithelial cells + - + Hematopoietic toxicity, Limited CAR-T persistence mRNA-based transient CAR-T to reduce long-term toxicity, Safety switch CAR-T to improve controllability [ 98 – 104 ] FRβ Folate-binding protein receptor Folate delivery Preclinical 70% of cases Myeloid cells - + + Low antigen density, Antigen escape ATRA to enhance antigen expression, Dual-target CAR-T to minimize resistance [ 105 – 109 ] LILRB4 Leukocyte Ig-like Receptor-B family Inhibitory receptor role in immune tolerance Preclinical / Myeloid antigen presenting - + + / / [ 110 – 116 ] WT1 Zinc-finger DNA binding protein Transcription factor Preclinical 80–90% of AML cases Kidney endometrium and testis cells - - + / / [ 117 – 120 ] PR1 Proteinase protein HLA-presented antigens Preclinical / Neutrophils - - + / / [ 121 , 122 ] CD4 Ig superfamily/Glycoprotein Transmembrane protein Preclinical 65.0% in M4, 78.3% in M5, and 30–40% in other AML subtypes T cells and myeloid cells - + + / / [ 123 – 125 ] CD47 Ig superfamily/Glycoprotein Binds SIRPa and inhibits cytophage Preclinical / Keratinocytes and immune cells + + + / / [ 126 – 131 ] CD96 Ig superfamily/Glycoprotein Adhesion of activated T and NK cells Preclinical / T, NK, and endothelial cells + + + / / [ 132 – 134 ] CD13 Zinc-dependent metalloprotease protein Aminopeptidase, transmembrane protein Preclinical 66–82% of cases Myeloid, epithelial, endothelial cells, and fibroblasts - + + / / [ 84 , 135 , 136 ] Abbreviations: AML, Acute myeloid leukemia; HSCs, Hematopoietic stem cells; LSCs, Leukemic stem cells; IL, Interleukin; DC, Dendritic cell; SIGLEC, Sialic acid-binding immunoglobulin-like lectin; Ig, Immunoglobulin; NK, Natural killer cell; CLEC12A, C-type lectin domain family 12 member A; NKG2DL, Natural killer group ligands; MICA, MHC class I polypeptide-related sequence A; MICB, MHC class I polypeptide-related sequence B; ULBP1-6, UL16-binding proteins; FLT3, Fms-like tyrosine kinase 3; ADP, Adenosine diphosphate; TNF, Tumor necrosis factor; Ley, Lewis Y; TIM3, T-cell immunoglobulin and mucin-domain containing-3; FRβ, Folate receptor beta; ADGRE2, Adhesion G protein-coupled receptor E2; SIGLEC-6, Sialic acid-binding immunoglobulin-like lectin 6; LILRB4, Leukocyte immunoglobulin-like receptor subfamily B member 4; WT1, Wilms’ Tumor 1; PR1, Proteinase 1; HLA, Human leukocyte antigen; SIRPa, Signal regulatory protein alpha

Table 2 Clinical outcomes of CAR-T therapy in AML Clinical trial ID Disease Type Target CAR-T Product Type Center Phase Estimated Participants Clinical Outcomes Adverse Events Research Institution Authors NCT04230265 r/r AML CD123 UniCAR-T-CD123 / I 8 screened, 5 apheresis, 3 treated ORR 100% (3/3: PR 1, CRi 2) CRS Grade 1: 67%, CRS Grade ≥ 2: 0%, no ICANS, no dose-limiting toxicities University Hospital Würzburg, Technical University Dresden, Comprehensive Cancer Center Mainfranken Wermke et al.[ 21 ] NCT05105152 AML CD33 DARIC33 (Rapamycin-regulated CAR-T) / I / / / Seattle Children’s Hospital, Seattle Children’s Therapeutics, Fred Hutchinson Cancer Center Appelbaum et al.[ 27 ] NCT03126864 r/r AML CD33 Autologous CD33-CAR-T cells Single-center I 10 enrolled, 3 treated ORR 0% (no CR or PR), death 100% (3/3) CRS Grade 2–3: 2 patients, ICANS Grade 2: 1 patient, tumor lysis syndrome with acute kidney injury Grade 3: 1 patient, increased ALT/AST Grade 3: 1 patient The University of Texas MD Anderson Cancer Center, Azienda Ospedaliera di Rilievo Nazionale Santobono-Pausilipon Tambaro et al.[ 25 ] NCT03222674 r/r AML (pediatric) CLL-1 autologous CLL-1 CAR-T cells Single-center I/II 4 enrolled, 4 treated ORR 100% (CR 75%, CRi 25%) CRS Grade 1–2: 100%, CRS Grade ≥ 3: 0%, no ICANS, no dose-limiting toxicities or treatment-related deaths Guangzhou Women and Children’s Medical Center, Shenzhen Geno-immune Medical Institute Zhang et al.[ 35 ] ChiCTR2000041054 r/r AML CLL-1 CLL-1 CAR-T cells / I 10 ORR 70% (CR/CRi 7/10), death 30% (3/10) CRS low-grade: 40%, CRS high-grade: 60%, no CRES, severe pancytopenia 100% (Grade 3/4 agranulocytosis 90%, anemia 70%, thrombocytopenia 70%) Tianjin First Central Hospital, The First Affiliated Central Hospital of Nankai University Jin et al.[ 33 ] NCT02203825 AML/MDS/MM NKG2DL Autologous NKG2D-CAR T cells Single-center I 12 Median OS 4.7 months, OS at 3 months 75%, OS at 6 months 42% No grade ≥ 3 AEs attributed to NKG2D-CAR T cells Dana-Farber Cancer Institute, Boston Children’s Hospital, Harvard Medical School Baumeister et al.[ 54 ] CTX 08 − 0002 r/r AML LeY Second-generation LeY CAR-T cells Single-center I 5 enrolled, 4 treated ORR 0% (no CR or PR), death 100% (4/4) no CRS, no ICANS, fever Grade 1/2: 50%, transient lymphopenia in all patients Peter MacCallum Cancer Centre, University of Melbourne Ritchie et al.[ 13 ] NCT04351022 AML (relapsed post-allo-HSCT) CD38 CD38-CAR-T (CAR-T-38) / / 6 ORR 66.7% (CR/CRi 4/6), median OS 7.9 months, OS at 6 months 50%, median LFS 6.4 months CRS Grade 1–2: 83.3%, CRS Grade 3 (hepatotoxicity): 16.7%, no ICANS, neutropenia (< 500/µl) 100%, thrombocytopenia (< 10,000/µl) 100%, infection 16.7% National Clinical Research Center for Hematologic Diseases, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University Cui et al.[ 67 ] Abbreviations: AML, Acute myeloid leukemia; CD33, Cluster of differentiation 33; CAR-T, Chimeric antigen receptor T-cell; OS, Overall survival; MDS, Myelodysplastic syndrome; MM, Multiple myeloma; NKG2DL, Natural killer group 2 member D ligand; ORR, Overall response rate; CR, Complete remission; CRi, Complete remission with incomplete hematologic recovery; LFS, Leukemia-free survival; PR, Partial remission; r/r, Relapsed/Refractory; CLL-1, C-type lectin-like molecule 1; LeY, Lewis Y; BM, Bone marrow

Table 3 Ongoing clinical trials of CAR-T therapy in AML Clinical trial ID Target Antigen AML Type Intervention Center Status Estimated participants Ages eligible Phase Study Start Study Complete Research Institution Location NCT04318678 CD123 CD123 + r/r AML Anti-CD123 CAR-T Multi-center Recruiting 32 Up to 21 Ⅰ 2020 2026 St. Jude Children’s Research Hospital 2 Academic medical centers across the United States NCT04272125 CD123 CD123 + r/r AML Anti-CD123 CAR-T Single-center Recruiting 40 3 to 75 Ⅰ/Ⅱ 2019 2024 Chongqing Precision Biotech Co., Ltd Chongqing, China NCT04265963 CD123 CD123 + r/r AML Anti-CD123 CAR-T Single-center Recruiting 45 2 to 75 Ⅰ/Ⅱ 2019 2024 Chongqing Precision Biotech Co., Ltd Kunming, Yunnan, China NCT04014881 CD123 CD123 + r/r AML Third-generation anti-CD123 CAR-T Single-center Unknown 50 18 to 70 Ⅰ 2019 2022 Wuhan Union Hospital Wuhan, Hubei, China NCT03556982 CD123 CD123 + r/r AML Anti-CD123 CAR-T Single-center Unknown 10 14 to 75 Ⅰ/Ⅱ 2018 2020 The Affiliated Hospital of the Chinese Academy of Military Medical Sciences Beijing, China NCT03585517 CD123 CD123 + r/r AML Anti-CD123 CAR-T Single-center Completed 10 3 to 80 Ⅰ 2018 2020 Beijing Immunochina Medical Science & Technology Co., Ltd. Beijing, China NCT03796390 CD123 CD123 + r/r AML Anti-CD123 CAR-T Single-center Unknown 15 2 to 65 Ⅰ 2018 2021 Hebei Senlang Biotechnology Inc., Ltd. Langfang, Hebei, China NCT03114670 CD123 CD123 + AML Anti-CD123 CAR-T Single-center Unknown 20 18 and up Ⅰ 2017 2021 Affiliated Hospital to Academy of Military Medical Sciences Beijing, China NCT04678336 CD123 r/r AML Anti-CD123 CAR-T Single-center Recruiting 18 1 to 29 Ⅰ 2021 2036 University of Pennsylvania Philadelphia, Pennsylvania, United States NCT03126864 CD33 CD33 + r/r AML Anti-CD33 CAR-T Single-center Terminated 11 1 to 80 Ⅰ 2017 2019 M.D. Anderson Cancer Center Houston, Texas, United States NCT06762132 CD33 CD33 + r/r AML Anti-CD33 CAR-T Single-center Recruiting 27 18 and up Ⅰ 2025 2027 The first affiliated hospital of medical college of Zhejiang University Hangzhou, Zhejiang, China NCT06326021 CD33 CD33 + r/r AML Anti-CD33 CAR-T Single-center Recruiting 27 1 to 70 Ⅰ 2024 2026 Beijing GoBroad Hospital Beijing, China NCT05945849 CD33 r/r AML Anti-CD33 CAR-T Single-center Recruiting 16 18 and up Ⅰ 2024 2044 University of Pennsylvania Philadelphia, Pennsylvania, United States NCT05672147 CD33 CD33 + r/r AML Anti-CD33 CAR-T Single-center Recruiting 27 18 and up Ⅰ 2023 2028 City of Hope Medical Center, United States Duarte, California, United States NCT05984199 CD33 CD33 + r/r AML Anti-CD34 CAR-T Multi-center Recruiting 24 18 and up Ⅰ/Ⅱ 2023 2026 Vor Biopharma 14 Academic medical centers across the United States NCT05473221 CD33 CD33 + r/r AML Anti-CD33 CAR-T Single-center Not yet recruiting 20 2 to 70 Ⅰ 2022 2025 The first affiliated hospital of medical college of Zhejiang University Hangzhou, Zhejiang, China NCT05445765 CD33 CD33 + AML Anti-CD33 CAR-T Single-center Unknown 10 2 to 60 Ⅰ 2022 2024 iCell Gene Therapeutics Langfang, Hebei, China NCT05105152 CD33 CD33 + r/r AML Anti-CD34 CAR-T Single-center Recruiting 18 Up to 30 Ⅰ 2021 2041 Seattle Children’s Hospital Seattle, Washington, United States NCT04835519 CD33 CD33 + r/r AML Anti-CD33 CAR-T Single-center Completed 5 1 to 70 Ⅰ/Ⅱ 2021 2023 Beijing Boren Hospital Beijing, China NCT03971799 CD33 CD33 + r/r AML Anti-CD34 CAR-T Multi-center Recruiting 52 1 to 35 Ⅰ/Ⅱ 2020 2039 Center for International Blood and Marrow Transplant Research 6 Academic medical centers across the United States NCT06128044 CLL-1 r/r AML a CRISPR-edited allogeneic anti-CLL-1 CAR-T Multi-center Recruiting 70 18 and up Ⅰ 2024 2027 Caribou Biosciences, Inc. 10 Academic medical centers across the United States NCT06017258 CLL-1 CLL1 + r/r AML Anti-CLL-1 CAR-T Multi-center Recruiting 15 1 and up Ⅰ 2023 2026 Memorial Sloan Kettering Cancer Center 7 Academic medical centers across the United States NCT05467202 CLL-1 CLL1 + r/r AML Anti-CLL-1 CAR-T Single-center Not yet recruiting 20 2 to 70 Ⅰ 2022 2025 The first affiliated hospital of medical college of Zhejiang University Hangzhou, Zhejiang, China NCT04219163 CLL-1 CLL1 + r/r AML Anti-CLL-1 CAR-T Single-center Recruiting 18 Up to 75 Ⅰ 2020 2038 Baylor College of Medicine Houston, Texas, United States NCT04884984 CLL-1 CLL1 + r/r AML Anti-CLL-1 CAR-T Single-center Unknown 20 6 to 65 Ⅰ/Ⅱ 2017 2024 The First Affiliated Hospital of Soochow University Suzhou, Jiangsu, China NCT06420063 CD123/CD33 CD33+/CD123+/CLL-1 + AML Dual-target CD123/CD33 CAR-T Single-center Recruiting 85 6 to 90 Ⅰ/Ⅱ 2024 2026 Essen Biotech Beijing, China NCT06125652 CD123/TIM3 r/r AML Dual-target CD123/TIM3 CAR-T Single-center Recruiting 20 18 to 70 Ⅰ/Ⅱ 2023 2027 Xuzhou Medical University Xuzhou, Jiangsu, China NCT03631576 CD123/​CLL-1 r/r AML Dual-target CD123/CLL-1 CAR-T Single-center Unknown 20 Up to 70 Ⅱ/Ⅲ 2018 2021 Fujian Medical University Union Hospital Fuzhou, Fujian, China NCT05654779 CD33/CLL-1 r/r AML Dual-target CD33/CLL-1 CAR-T Multi-center Terminated 4 14 to 60 Ⅰ 2022 2023 Institute of Hematology & Blood Diseases Hospital 2 Academic medical centers across China NCT05467254 CD33/CLL-1 CD33+/CLL-1 + r/r AML Dual-target CD33/CLL-1 CAR-T Single-center Not yet recruiting 20 2 to 70 Ⅰ 2022 2025 The first affiliated hospital of medical college of Zhejiang University Hangzhou, Zhejiang, China NCT05248685 CD33/CLL-1 CD33+/CLL-1 + r/r AML Dual-target CD33/CLL-1 CAR-T Single-center Unknown 20 1 to 70 Ⅰ 2022 2024 Beijing Boren Hospital Beijing, China NCT05016063 CD33/CLL-1 CD33 + AML Dual-target CD33/CLL-1 CAR-T Single-center Unknown 32 18 to 70 Ⅰ 2021 2023 Xinqiao Hospital, China Chongqing, China NCT04658004 NKG2DL NKG2DL + r/r AML Anti-NKG2DL CAR-T Single-center Not yet recruiting 36 3 to 70 Ⅰ 2021 2027 The first affiliated hospital of medical college of Zhejiang University Hangzhou, Zhejiang, China NCT06786533 FLT3 FLT3 + r/r AML Anti-FLT3 CAR-T Single-center Recruiting 18 18 and up Ⅰ 2025 2027 Hemogenyx Pharmaceuticals LLC Houston, Texas, United States NCT05445011 FLT3 FLT3 + r/r AML Anti-FLT3 CAR-T Single-center Recruiting 12 18 to 70 Ⅰ 2022 2027 Wuhan Union Hospital Wuhan, Hubei China NCT06760260 FLT3 FLT3 + r/r AML Anti-FLT3 CAR-T Single-center Recruiting 7 18 to 70 Ⅰ 2022 2026 Hrain Biotechnology Co., Ltd. Suzhou, Jiangsu, China NCT05432401 FLT3 FLT3 + r/r AML Anti-FLT3 CAR-T Single-center Recruiting 18 18 to 70 Ⅰ 2022 2025 PersonGen BioTherapeutics (Suzhou) Co., Ltd. Wuhan, Hubei China NCT05023707 FLT3 FLT3 + r/r AML Anti-FLT3 CAR-T Single-center Recruiting 5 16 to 65 Ⅰ/Ⅱ 2021 2025 The First Affiliated Hospital of Soochow University Suzhou, Jiangsu, China NCT05017883 FLT3 FLT3 + r/r AML Anti-FLT3 CAR-T Single-center Recruiting 5 18 to 70 / 2021 2025 PersonGen BioTherapeutics (Suzhou) Co., Ltd. Hefei, Anhui, China NCT04351022 CD38 CD38 + r/r AML Anti-CD38 CAR-T Single-center Unknown 20 6 to 65 Ⅰ/Ⅱ 2017 2023 The First Affiliated Hospital of Soochow University Suzhou, Jiangsu, China NCT05748197 ADGRE2 r/r AML ADCLEC.syn1 CAR T cells Multi-center Recruiting 40 18 and up Ⅰ 2024 2028 Memorial Sloan Kettering Cancer Center 7 Academic medical centers across the United States NCT05463640 ADGRE2 ADGRE2 + r/r AML Anti-ADGRE2 CAR-T Single-center Not yet recruiting 20 2 to 70 Ⅰ 2022 2025 The first affiliated hospital of medical college of Zhejiang University Hangzhou, Zhejiang, China NCT05488132 SIGLEC-6 r/r AML Anti-SIGLEC-6 CAR-T Single-center Recruiting 20 18 to 70 Ⅰ/Ⅱ 2022 2025 Xuzhou Medical University Xuzhou, Jiangsu, China Abbreviations: AML, Acute myeloid leukemia; CAR, Chimeric antigen receptor; r/r, Relapse/Refractory; TIM3, T-cell immunoglobulin and mucin-domain containing-3; CLL-1, C-type lectin-like molecule 1; NKG2DL, Natural killer group 2 member D ligands; FLT3, Fms-like tyrosine kinase 3; ADGRE2, Adhesion G protein-coupled receptor E2; SIGLEC-6, Sialic acid-binding Ig-like lectin 6

Early efforts evaluated folate receptor beta (FRβ) and the carbohydrate antigen Lewis Y (LeY) as potential targets for CAR-T therapy in AML. Preclinical models showed that FRβ CAR-T cells exhibited selective cytotoxicity against leukemic blasts while sparing hematopoietic stem cells, although mature myeloid cells were also depleted [ 105 – 107 ]. Early-phase trials of LeY CAR-T therapy demonstrated transient clinical remissions, with engineered T cells detectable in circulation for several months in some patients [ 89 , 90 ]. However, durable responses were not achieved. LeY CAR-T cells showed limited long-term persistence in vivo [ 89 ], whereas FRβ-directed approaches, despite potent anti-leukemic activity, also recognized normal myeloid populations, raising concerns about on-target toxicity [ 105 ]. These targets now serve as historical benchmarks, guiding refinements in antigen selection and safety profiling.

CD123

CD123, the α subunit of the interleukin-3 receptor (IL-3Rα), is overexpressed on > 80% of AML blasts and LSCs, where it mediates proliferative and anti-apoptotic signaling through the JAK/STAT, PI3K/AKT, and MAPK pathways [ 10 – 12 ]. High CD123 expression is frequently observed in AML subtypes harboring FLT3-ITD and NPM1 mutations, with evidence indicating transcriptional upregulation driven by these alterations [ 10 ]. Perriello et al. further confirmed uniform CD123 expression on NPM1-mutated AML, particularly within CD34⁺CD38⁻ compartments, supporting its potential as a lineage marker for immunotherapeutic targeting in this molecular subset [ 13 ]. In addition, Aref et al. identified that co-expression of CD123 and CD25 was associated with a prognostically adverse group with significantly lower induction remission rates and shorter overall survival, highlighting its utility in risk stratification beyond cytogenetics [ 11 ].

Due to its high and consistent expression on AML blasts and leukemia stem cells, CD123 has been extensively evaluated as a target for CAR-T cell therapy. In early studies, CD123 CAR-T cells containing a CD28/CD3ζ signaling module were generated [ 14 ]. Lysis of CD123⁺ AML cell lines (KG1a) and primary AML blasts was observed in vitro, while colony formation of normal granulocyte–macrophage and erythroid progenitors remained largely preserved [ 14 ]. In a NOD/Scid xenograft model of systemic AML, a single infusion of these CAR-T cells reduced tumor burden and extended survival compared with untreated controls [ 14 ]. A subsequent study evaluated a 4-1BB/CD3ζ–based construct in MOLM14 cells and primary AML samples [ 15 ]. In NSG and NSGS mouse models, leukemic cells were fully eradicated, and survival benefits were maintained regardless of baseline CD123 expression levels [ 15 ].

Baroni and colleagues further investigated this toxicity by comparing CD28- and 4-1BB–costimulated CD123 CAR-T cells in humanized hematopoietic models [ 10 ]. NSG mice engrafted with human umbilical cord blood CD34⁺ cells received either CAR-T design, and both completely eliminated hematopoietic progenitors, resulting in loss of multilineage bone marrow reconstitution [ 10 ]. To mitigate these risks, a modular UniCAR-T system was developed. In a first-in-human phase I trial ( NCT04230265 ), patients with relapsed or refractory (R/R) AML received autologous UniCAR-T cells following fludarabine/cyclophosphamide lymphodepletion and continuous infusion of a soluble CD123-specific targeting module (TM123) for 25–29 days [ 16 ]. Among the initial three patients, two achieved complete remission with incomplete hematologic recovery and one achieved partial remission [ 16 ]. Cytokine release syndrome (CRS) was limited to grade 1, and hematopoietic suppression resolved rapidly after TM123 withdrawal [ 16 ]. Persistence of UniCAR-T cells was documented for up to six months, allowing re-administration of TM123 in one patient [ 16 ]. Additional phase I/II clinical trials (e.g., NCT04318678 , NCT04272125 , NCT04265963 ) are ongoing to further assess the safety, response rates, and durability of CD123 CAR-T therapy in AML (Table 3 ).

CD33

CD33 (Siglec-3) is one of the most broadly expressed myeloid antigens in AML. Approximately 90% of AML blasts and LSCs display high levels of CD33, while partial expression is also observed in normal myeloid progenitors [ 17 ]. Elevated CD33 expression is frequently associated with NPM1 and FLT3 mutations, indicating a significant role in leukemogenesis and disease relapse [ 18 ].

The clinical feasibility of targeting CD33 has been demonstrated by antibody–drug conjugates such as gemtuzumab ozogamicin (GO) [ 17 ]. In preclinical studies, CAR-T cells engineered with a GO-derived single-chain variable fragment (CART33) showed potent antileukemic activity, effectively eradicating AML cells in MOLM14 and MV4;11 cell lines as well as in NSG mouse xenograft models [ 19 ]. Early-phase clinical testing of autologous CD33 CAR-T cells ( NCT03126864 ) enrolled ten patients with R/R AML [ 20 ]. CAR-T products meeting release criteria were successfully manufactured for three patients [ 20 ]. One patient achieved morphological remission, but relapse occurred nine weeks post-infusion. Grade 3 CRS was the primary treatment-related toxicity, and the overall feasibility of product manufacturing remained limited [ 20 ].

More recently, Qin and colleagues performed a systematic preclinical evaluation of six CD33-CAR constructs. The lintuzumab-based CD28/CD3ζ CAR exhibited the strongest antileukemic efficacy, both in AML cell lines and in pediatric patient-derived xenograft (PDX) models, and has been selected for clinical translation [ 21 ]. Furthermore, Appelbaum et al. developed a drug-regulatable CAR platform (DARIC33), enabling rapamycin-mediated control of CAR activity [ 22 ]. This approach achieved efficient and reversible leukemia clearance in MOLM14 and THP-1 xenograft models and has advanced to a phase I clinical trial ( NCT05105152 ) [ 22 ]. Several additional CD33 CAR-T trials are currently underway across multiple centers (Table 3 ).

CLEC12A (CLL-1)

CLEC12A (CLL-1), a myeloid-specific transmembrane glycoprotein, is predominantly expressed in AML blasts and LSCs but minimally in HSCs, representing a favorable CAR-T target with limited hematopoietic toxicity [ 23 ]. Its stable expression throughout AML progression further supports its utility in MRD detection and immunotherapy [ 24 ]. Minimal expression in lymphoid compartments further reduces the risk of B- and T-cell immune reconstitution impairment [ 25 ]. Nonetheless, bimodal antigen expression and low CLEC12A density observed in certain patient subsets have been associated with suboptimal remission outcomes, underscoring antigen heterogeneity as a therapeutic challenge [ 26 ].

Preclinical studies have demonstrated selective and potent antileukemic activity of CLL-1 CAR-T cells. Wang et al. designed a CAR construct with CD28, 4-1BB, and CD3ζ signaling domains that effectively eliminated CLL-1⁺ AML cells and myeloid progenitors while sparing CLL-1⁻ HSCs [ 25 ]. In NSG mouse xenograft models, these CAR-T cells significantly reduced leukemic burden and extended survival [ 25 ]. Further optimization was achieved by Ataca et al., who introduced IL-15 co-expression via a dual-vector system [ 27 ]. The modified CAR-T cells exhibited more than a tenfold increase in proliferation ( p < 0.0001), maintained a central memory phenotype, and achieved complete disease clearance in MOLM-14 and PDX models [ 27 ]. However, elevated TNFα, IL-2, and IL-15 secretion induced acute inflammatory toxicities in lung and liver tissues, which were mitigated by TNFα blockade or activation of an inducible caspase-9 safety switch [ 27 ].

Clinical evidence has further validated the therapeutic potential of CLL-1 CAR-T therapy. In a first-in-human Phase I trial, autologous CAR-T infusion in ten adults with R/R AML achieved a 70% complete remission or incomplete hematologic recovery rate [ 28 ]. All patients experienced CRS, 60% developed grade ≥ 3 CRS, and two died from infection secondary to prolonged granulocytopenia, suggesting that hematopoietic stem cell transplantation may be necessary for hematologic recovery [ 29 ]. In a pediatric Phase I/II trial, four children received autologous CLL-1 CAR-T cells incorporating an inducible FKBP–caspase-9 safety switch; three achieved MRD-negative complete remission, and one survived over five months, with only mild and manageable adverse events [ 30 ]. Given the small cohort size of published trials, ongoing early-phase clinical studies (Table 3 ) are currently evaluating dosing regimens, safety, and long-term outcomes of CLL-1 CAR-T therapy in both adult and pediatric AML populations.

CD70

CD70, a type II transmembrane glycoprotein belonging to the tumor necrosis factor ligand (TNF) family, is the sole ligand for CD27 and regulates immune cell survival and activation [ 137 ]. Normally transiently expressed on activated immune cells, CD70 is aberrantly upregulated in AML blasts and LSCs, driving immune evasion, proliferation, and chemoresistance [ 64 ]. Expression levels vary considerably among patients, with a subset displaying low antigen density below flow cytometric detection thresholds [ 65 ]. Nevertheless, CD70 remains a selective immunotherapeutic target due to its minimal expression on normal HSCs and its potential inducibility through epigenetic modulation, which may enhance susceptibility to targeted therapy [ 66 ]. Wu et al. demonstrated potent cytotoxicity of CD70 CAR-T cells against CD70⁺ AML cell lines, including THP-1, MOLM-13, OCI-AML3, and MV4-11, as well as primary patient blasts, with robust cytokine production and proliferative capacity [ 66 ]. In a MOLM-13 xenograft model, CD70 CAR-T cells significantly reduced tumor burden and prolonged survival, although residual leukemic cells persisted [ 66 ]. Additional engineering strategies, including HLA-independent TCR receptors, achieved superior antigen sensitivity and improved tumor control in xenograft models compared to conventional CAR-T cells [ 138 ]. However, clinical translation of CD70 CAR-T cells remains at a preclinical stage, and its therapeutic window will require further validation in human models.

FLT3

FLT3, a type III receptor tyrosine kinase, regulates hematopoietic homeostasis in progenitor cells [ 40 ]. However, FLT3-ITD, present in ~ 30% of AML cases, drives leukemogenesis through PI3K/Akt, Raf/MEK/ERK, and JAK/STAT5 activation, enhancing proliferation and survival while inhibiting apoptosis [ 41 , 137 ]. Despite the approval of FLT3 inhibitors (e.g., midostaurin, gilteritinib), acquired resistance limits their long-term efficacy [ 41 – 43 ]. These challenges have driven the exploration of FLT3-targeted immunotherapies with enhanced specificity and sustained efficacy.

A CAR-T platform utilizing the native ligand FLT3 ligand (Flt3Lg) as the antigen recognition domain has been developed by Maiorova et al., offering a strategy that minimizes spontaneous activation and reduces background signaling compared to conventional scFv-based CARs [ 44 ]. This FLT3 CAR selectively eliminates FLT3 + AML cell lines (e.g., MOLM13, MV4-11) and primary patient-derived leukemic blasts in vitro, without inducing cytotoxicity in FLT3 − normal cells such as peripheral blood mononuclear cells, B cells, or T cells [ 44 ]. In xenograft models, it markedly reduced leukemic burden while preserving normal myeloid compartments [ 44 ]. To further improve safety, a rationally designed L27P point mutation was introduced into the Flt3Lg domain, generating a modified construct (Flt3m-CAR) that limits receptor recycling and attenuates STAT5 phosphorylation without compromising antigen specificity [ 45 ]. This mutated CAR demonstrated reduced hematopoietic suppression and improved tolerability in preclinical models, illustrating a structural optimization strategy for ligand-based CAR design [ 45 ]. Building on these preclinical advances, multiple clinical trials are now actively recruiting patients with FLT3⁺ AML to evaluate the safety and efficacy of FLT3 CAR-T therapy (Table 3 ). However, current clinical evidence remains limited to small, early-phase, single-center studies, and further validation in larger, multi-center cohorts is required to determine real-world feasibility and durability of response.

NKG2D ligands

NKG2D is a co-stimulatory receptor expressed on NK cells, CD8⁺ T cells, and γδ T cells, mediating cytotoxicity through DAP10 signaling upon engagement with multiple stress-induced ligands, including MICA, MICB, and members of the ULBP family [ 70 – 72 ]. In AML, higher expression of NKG2D ligands (NKG2DLs) has been correlated with improved immune clearance and patient survival [ 71 , 73 ]. However, the absence of NKG2DLs on LSCs facilitates immune evasion and contributes to disease persistence [ 74 ]. In a patient-derived xenograft model, NKG2DL⁻ LSCs demonstrated robust self-renewal and resistance to both CAR-T and NK cell–mediated cytotoxicity [ 74 ]. Several immune evasion mechanisms further limit its therapeutic utility. AML cells actively shed soluble NKG2DLs through protease-mediated cleavage, leading to internalization of the CAR receptor and exhaustion of effector cells [ 70 , 75 ].

Preclinical studies support the feasibility of targeting NKG2DL through NKG2D CAR-T cells. The construct developed by Driouk et al. incorporates the full-length NKG2D extracellular domain, enabling recognition of multiple ligand subtypes [ 76 ]. These CAR-T cells exhibited potent cytotoxicity against AML cell lines including HL-60, OCI-AML3, and THP-1, and delayed leukemia progression in xenograft models using NSG mice [ 76 ]. Importantly, cytolytic activity was preserved despite low ligand density, likely reflecting the broad ligand recognition profile of NKG2D [ 76 ]. In clinical settings, Baumeister et al. conducted a Phase I trial ( NCT02203825 ) involving autologous NKG2D CAR-T cells in patients with AML, myelodysplastic syndromes, or multiple myeloma [ 77 ]. The treatment demonstrated a favorable safety profile, with no dose-limiting toxicities or severe immune-related adverse events. Grade 1 CRS was observed in several patients, and no neurotoxicity occurred [ 77 ]. However, objective responses were not achieved, and only transient disease stabilization was observed [ 77 ].

TIM3

TIM-3, a type I transmembrane protein initially identified on CD4⁺ Th1 and CD8⁺ Tc1 cells, is also expressed on NK cells and dendritic cells and is now recognized as highly enriched on CD34⁺CD38⁻ LSCs in AML [ 79 – 82 , 139 ]. Its ligand interaction with Galectin-9 activates the HCK–p120-catenin cascade, triggering non-canonical Wnt/β-catenin signaling, enhancing LSC self-renewal and upregulating c-Myc and BCL-2, thereby promoting chemoresistance [ 83 ]. Genetic ablation of TIM-3 reduced LSC colony formation and leukemic burden in murine models [ 83 ]. Clinically, TIM-3⁺ LSCs persist post-induction and associate with relapse risk, poor remission, and elevated peripheral leukocytes [ 80 , 139 ]. This subpopulation is detectable in approximately 57% of newly diagnosed AML patients [ 139 ]. Notably, TIM-3 is significantly overexpressed in AML harboring core-binding factor fusion genes such as t(8;21) and inv(16), suggesting its utility as a molecular stratification marker [ 82 ]. Co-expression of TIM-3 with immunosuppressive molecules, including PD-L1 and Galectin-9, further implicates it in shaping an immune-evasive microenvironment [ 81 ].

Several TIM-3 CAR-T cell constructs demonstrated potent anti-leukemic efficacy in AML PDX models [ 84 ]. However, off-target cytotoxicity against normal CD34⁺ HSCs limited their therapeutic window [ 84 ]. To address this issue, a dual-signal “Split CAR” strategy was developed, employing TIM-3 as the recognition domain and CD13 as the activation domain. T-cell cytotoxicity is initiated only upon co-recognition of both antigens, preserving anti-leukemic activity while minimizing hematopoietic toxicity [ 84 ]. No cumulative toxicity was observed in second-generation humanized transplantation models [ 84 ]. Additionally, iPSC-derived TIM-3 CAR-NK cells have been developed and demonstrated cytotoxic efficacy in AML models, supporting the broader feasibility of combining novel CAR targets with universal NK cell platforms [ 85 ]. Despite robust preclinical efficacy, TIM-3 CAR-based therapies have not yet progressed to clinical trials.

CD38

CD38, a class II transmembrane glycoprotein, functions as both an NAD⁺-metabolizing enzyme and a cell adhesion receptor [ 31 ]. It is broadly expressed across the hematopoietic system, with particularly high expression in multiple myeloma, where it has been successfully targeted by monoclonal antibodies such as daratumumab [ 32 , 33 ]. In AML, CD38 expression is heterogeneous but remains elevated in the majority of leukemic blasts, suggesting its suitability as an immunotherapeutic target [ 32 , 33 ]. However, its limited expression on LSCs may restrict long-term efficacy [ 32 , 33 ]. The therapeutic potential of CD38 CAR-T cells has been explored in preclinical and clinical settings. Robust cytolytic activity has been demonstrated in AML cell lines with high CD38 expression (e.g., THP-1, CMK), whereas cell lines with low antigen density (e.g., HL-60, U937) show markedly reduced sensitivity, emphasizing the importance of target abundance in determining efficacy [ 34 ]. A phase I clinical trial ( NCT04351022 ) reported complete remission (CR) or CR with incomplete hematologic recovery (CRi) in four of six patients (66.7%) with relapsed AML, with a six-month leukemia-free survival (LFS) rate of 50% [ 35 ]. Flow cytometry confirmed selective depletion of CD38⁺ leukemic cells with no significant evidence of off-tumor cytotoxicity [ 35 ]. Despite these encouraging results, CD38 CAR-T therapy faces notable barriers. Endogenous CD38 expression in engineered T cells may trigger fratricide, thereby compromising their expansion, persistence, and therapeutic potency in vivo [ 31 , 36 ].

CD44v6

CD44v6, a splice isoform of the CD44 adhesion molecule, is aberrantly expressed in several malignancies, including AML, where it contributes to leukemogenesis by promoting cell adhesion, migration, and resistance to chemotherapy [ 37 – 39 ]. As a marker associated with LSCs, its expression has been correlated with poor prognosis and disease persistence [ 37 – 39 ]. Recent studies have identified CD44v6 as a mutation-associated surface antigen enriched in AML subsets harboring FLT3-ITD or DNMT3A-R882H mutations [ 39 ]. In isogenic SKM-1 cells engineered to express these mutations, CD44v6 expression was significantly upregulated, rendering cells more susceptible to CD44v6 CAR-T cell cytotoxicity [ 39 ]. This upregulation was driven by promoter hypomethylation, as confirmed through methylation profiling and luciferase reporter assays, establishing a direct epigenetic link between oncogenic signaling and antigen expression [ 39 ]. Building upon this mechanistic rationale, Casucci et al. developed second-generation CD44v6 CAR-T cells incorporating a CD28 costimulatory domain and a suicide switch (either HSV-TK or inducible caspase-9) [ 37 ]. These engineered cells exhibited potent cytotoxicity against AML and multiple myeloma cells, including MOLM-13 and RPMI-8226, and achieved complete tumor eradication in xenograft models [ 37 ]. Notably, minimal off-tumor toxicity was observed: CD34⁺ hematopoietic stem cells and keratinocytes were spared, and transient monocytopenia represented the only detectable adverse event [ 37 ]. The suicide system allowed pharmacologic elimination of CAR-T cells when needed, providing an additional safety mechanism [ 37 ].

CD117

CD117 (c-KIT) is a receptor tyrosine kinase primarily expressed on HSCs and progenitor cells [ 98 ]. In AML, CD117 is detected in approximately 85% of patients [ 99 ], with a particularly high prevalence in the core-binding factor AML subtype [ 99 , 100 ], where alterations in CD117 signaling contribute to poor prognosis. Given its critical role in AML cell survival and proliferation, CD117 represents a promising therapeutic target [ 98 ]. CD117 CAR-T cells exhibit potent anti-leukemic activity, effectively depleting CD117⁺ AML cells in vitro and in vivo [ 98 , 101 ]. To address these challenges, researchers have explored multiple approaches to improve the safety of CD117 CAR-T therapy. Transient CAR expression via mRNA electroporation enables rapid attenuation post-AML clearance, minimizing prolonged hematopoietic toxicity [ 98 ]. Alternatively, the Sleeping Beauty transposon system combined with an inducible Caspase-9 safety switch allows selective CAR-T elimination upon drug administration, facilitating subsequent allo-HSCT and ensuring hematopoietic reconstitution [ 98 ]. Rituximab combined with ATG facilitates CAR-T depletion in later treatment phases, mitigating long-term persistence and associated risks [ 101 ].

CD13

CD13, or aminopeptidase N, is a zinc-dependent transmembrane protease abundantly expressed in myeloid progenitor cells, monocytes, and various solid tumors [ 84 ]. Its upregulation in AML and leukemic stem cells prompted the development of CD13-directed CAR-T strategies. Using the Sequential Tumor-Selected Antibody and Antigen Retrieval (STAR) platform, He et al. identified the nanobody Nb157 and generated CD13 CAR-T cells [ 84 ]. These CAR-T cells exhibited potent cytotoxic activity against AML cell lines, including HL-60 and KG-1, and achieved effective disease control in NSG mouse xenograft models engrafted with primary AML blasts [ 84 ]. Despite this efficacy, significant cytotoxicity toward normal HSPCs was observed, resulting in myelosuppression in preclinical models [ 84 ]. To mitigate this toxicity, a switchable CAR system incorporating nanobody-based activation control was subsequently engineered [ 135 ]. In this design, CAR-T cells remain inactive unless triggered by an exogenous antibody switch, allowing for reversible and dose-titratable activation [ 135 ]. At higher switch concentrations, AML cells were effectively eliminated; at reduced doses, cytotoxic effects on HSPCs were markedly attenuated while preserving anti-leukemic function [ 135 ].

ADGRE2 (EMR2)

ADGRE2 (also known as EMR2) is selectively overexpressed in AML, particularly within LSCs, while its expression remains minimal in normal HSPCs [ 93 ]. Single-cell RNA sequencing and flow cytometry analyses have confirmed its enrichment in relapse-associated clones, where it co-localizes with LSC markers such as CD96 and CD123 [ 140 ]. Functionally, ADGRE2 facilitates leukemogenesis by activating the PLCβ–PKC–MEK–ERK signaling cascade and regulating AP1 and DUSP1 expression, thereby sustaining proteostasis and promoting apoptotic resistance [ 93 ]. In addition, ADGRE2^high AML cells exhibit impaired susceptibility to NK and T cell–mediated immune clearance [ 140 ].

To leverage this LSC-restricted expression profile while minimizing off-leukemia toxicity, a dual-receptor CAR system—termed ADCLEC.syn1—was developed. This construct combines an ADGRE2-targeting CAR with a CLEC12A-directed chimeric co-stimulatory receptor (CCR), designed to operate via an IF-BETTER Boolean logic gating mechanism. CLEC12A-CCR alone lacks cytotoxic function but delivers selective 4-1BB costimulatory signaling to the ADGRE2 CAR when both antigens are co-expressed [ 140 ]. This configuration enables preferential activation in ADGRE2^highCLEC12A^+ AML cells, while sparing normal myeloid cells expressing only one or low levels of the target antigens [ 140 ]. Preclinical studies in NSG mouse models engrafted with primary human AML samples demonstrated that ADCLEC.syn1 CAR-T cells eliminate leukemic cells and significantly extend survival relative to single-input CARs [ 140 ]. Furthermore, the preservation of multilineage hematopoiesis in human CD34⁺ HSPC-engrafted mice indicates a favorable safety profile with reduced hematopoietic toxicity [ 140 ]. These findings support the translational potential of ADGRE2-directed combinatorial CAR designs, with a phase I clinical trial of ADCLEC.syn1 currently underway ( NCT05748197 ).

SIGLEC-6

Siglec-6, a member of the sialic acid-binding immunoglobulin-like lectin family, exhibits highly restricted expression under physiological conditions. Its presence is largely confined to placental trophoblasts, mast cells, and specific subsets of memory B cells, with negligible expression in HSPCs [ 46 ]. In AML, Siglec-6 is markedly upregulated, particularly within LSC-enriched subpopulations, where it frequently co-localizes with stemness-associated markers such as CD96 and CD123 [ 46 ]. Based on these features, Jetani et al. engineered a second-generation CAR construct incorporating a fully human Siglec-6–specific scFv linked to 4-1BB and CD3ζ signaling domains [ 46 ]. These CAR-T cells demonstrated potent and selective cytotoxicity against primary AML blasts and LSC-enriched fractions ex vivo, while sparing normal CD34⁺ HSPCs [ 46 ]. In NSG mice engrafted with primary human AML cells, Siglec-6 CAR-T therapy induced durable leukemia clearance and significantly prolonged survival [ 46 ]. No hematopoietic toxicity was detected, as evidenced by preserved multilineage hematopoietic reconstitution from co-transplanted human CD34⁺ cells [ 46 ]. Moreover, these CAR-T cells also exhibited activity against Siglec-6⁺ CLL samples, suggesting broader applicability across hematologic malignancies [ 46 ].

UMG1⁺ CD43 glycoform

Glycosylation represents a key immune evasion strategy in hematologic malignancies and is increasingly recognized as a promising axis for next-generation immunotherapeutic interventions [ 141 , 142 ]. Compared with conventional protein antigens, cancer-associated glycoforms exhibit enhanced specificity for malignant cells and may improve the safety profile of CAR-T therapy by minimizing off-target effects on normal HSPCs [ 141 , 143 ]. Among these, the sialylated isoform of CD43 (CD43s) emerges as a compelling candidate. CD43s is consistently upregulated across diverse leukemic subsets while remaining largely undetectable in healthy CD34⁺ HSPCs [ 143 ]. Its expression correlates with aberrant glycosyltransferase activity, particularly involving altered O-glycan elongation and terminal sialylation, suggesting that CD43s reflects a tumor-specific glycosylation phenotype [ 143 , 144 ].

Therapeutic targeting of CD43s has been explored using UMG1, a monoclonal antibody recognizing a conformation-dependent epitope (INEGSPLW) that requires O-glycan integrity [ 145 ]. This glycoepitope is selectively expressed on malignant T cells and subsets of AML cells but is absent in normal T cells and HSPCs [ 145 ]. A bispecific T-cell engager (BTCE) derived from UMG1 demonstrated potent cytotoxic activity in preclinical models of T-cell acute lymphoblastic leukemia (T-ALL) [ 145 ]. In Jurkat and HPB-ALL cell lines, UMG1-BTCE induced T cell–mediated lysis accompanied by increased secretion of IFN-γ and TNF-α [ 145 ]. In NSG xenograft mice, the construct eradicated T-ALL tumors and significantly prolonged survival without evident toxicity to peripheral T cells or CD34⁺ hematopoietic progenitors [ 145 ]. These findings validate CD43s as a glycosylation-restricted tumor-associated epitope and support the potential use of UMG1-derived scFvs in CAR architecture. Although UMG1-based CAR-T constructs have not yet been reported, additional preclinical studies reinforce the therapeutic relevance of CD43s targeting in AML. For example, the anti-CD43s antibody AT1413 significantly inhibited AML progression in NSG models without impairing normal hematopoietic progenitor function [ 146 ].

Translational challenges in advancing CAR-T therapy for AML

Biological constraints in target selection for AML

AML fundamentally challenges the development and application of CAR-T therapy due to the nature of its cellular origin and the extensive antigenic overlap with the normal hematopoietic compartment [ 1 , 147 , 148 ]. In contrast to B-ALL, which arises from lineage-committed B-cell precursors and expresses the well-defined antigen CD19, AML typically originates from HSCs or multipotent progenitor cells [ 1 ]. These populations share surface antigen profiles with normal myeloid progenitors, and no truly AML-specific antigen has been identified to date [ 149 ]. This biological overlap imposes major limitations on target selection for CAR-T therapy. In B-ALL, CAR-T cells eliminate both malignant and normal CD19⁺ B cells, with resultant B-cell aplasia being manageable through immunoglobulin replacement—representing a model of reversible toxicity [ 150 ]. In contrast, the leading AML-associated antigens such as CD33 and CD123 are also broadly expressed on essential normal myeloid progenitors [ 151 ]. Targeting these molecules with conventional CAR-T constructs results in a profound myeloablative effect, often leading to irreversible bone marrow failure [ 1 ]. In this context, the sustained persistence of lentiviral CAR-T cells—which enhances therapeutic durability in lymphoid malignancies—becomes a liability due to prolonged myelotoxicity. These challenges necessitate a conceptual shift in the therapeutic strategy. Rather than a model of selective cytotoxicity, AML-directed CAR-T therapy must be envisioned as a “clearance-and-reconstitution” paradigm, in which CAR-T–mediated ablation of both malignant and healthy myeloid cells is followed by allo-HSCT to restore hematopoiesis [ 1 ]. Beyond antigenic overlap, AML exhibits extensive phenotypic and genetic heterogeneity, both across patients and within individual cases [ 1 , 147 , 148 ]. The dynamic evolution of leukemic subclones leads to temporal and spatial variability in antigen expression, further complicating target selection [ 1 ]. Broadly expressed targets may cover a larger patient population but are often associated with unacceptable toxicity [ 148 ].

High tumor burden

Patients with AML typically present with a substantial leukemic burden at diagnosis [ 152 , 153 ]. Extensive infiltration of blasts throughout the bone marrow and peripheral blood establishes a microenvironment with markedly elevated antigen density. This heightened antigen load promotes robust CAR-T cell activation and increases the risk of CRS and other immunotoxicities [ 150 , 153 ]. Although systematic data remain limited, early studies suggest a positive correlation between blast percentage and CRS severity, with patients harboring ≥ 25% blasts exhibiting higher rates of grade ≥ 3 CRS [ 1 , 153 ]. To mitigate this risk, cytoreductive strategies such as chemotherapy or leukemic debulking are frequently employed [ 147 , 154 ]. However, these interventions may impair hematopoietic reserve and further delay marrow recovery [ 151 ]. Notably, CAR-T–related toxicities often occur in a profoundly myelosuppressed setting, either due to prior cytotoxic treatment or disease-related marrow failure, thereby complicating infection control and supportive care [ 148 , 153 ].

Barriers in CAR-T cell manufacturing and application

Timing constraints and the infeasibility of Pre-treatment T-Cell collection

AML progresses rapidly, with a median survival of only a few weeks to several months if left untreated [ 155 ]. Induction chemotherapy is typically initiated immediately after diagnosis, particularly in patients with hyperleukocytosis [ 156 ]. In a contemporary single-center cohort of 187 patients, the median diagnosis-to-treatment time (TDT) was 5.4 days, and 70% began chemotherapy within 1–5 days. Initiation ≥ 11 days after diagnosis was associated with a significantly lower complete remission rate [ 156 ]. Similarly, a multicenter analysis of 1,317 younger patients (≤ 60 years) found that a ≥ 5-day delay in treatment reduced the CR rate (59% vs. 73%, P = 0.008) and worsened overall survival (median 12.3 vs. 16.1 months; HR = 1.27, P = 0.03) [ 157 ]. An analysis of 55,985 AML cases from the U.S. National Cancer Database further confirmed that, in patients under 60 years, a ≥ 5-day delay in chemotherapy initiation was linked to poorer long-term survival (HR = 1.05, 95% CI: 1.01–1.09) [ 158 ]. These treatment timelines make it rarely feasible to perform leukapheresis within the short interval available to harvest sufficient autologous T cells for CAR-T manufacture.

T cell dysfunction in AML

At diagnosis, T cells from patients with AML display marked phenotypic and functional impairments. Peripheral counts are often preserved or even elevated compared with healthy donors, yet stimulation assays consistently demonstrate reduced secretion of IFN-γ, TNF-α, and IL-2 [ 147 , 159 ]. Flow cytometric analysis reveals increased PD-1⁺ frequencies (33.83% vs. 25.16% in controls), with a larger difference among CD8⁺ cells (11.81% vs. 6.62%) [ 160 ]. TIM-3 expression is elevated on both CD4⁺ and CD8⁺ subsets, and PD-1/TIM-3 co-expression is enriched within the bone marrow compartment [ 161 ]. High PD-1 expression on CD8⁺ cells and elevated TIM-3 on CD4⁺ cells independently predict shorter relapse-free and event-free survival, whereas greater marrow co-positivity correlates with lower remission rates after initial induction therapy [ 162 ]. Single-cell RNA sequencing further identifies depletion of central and effector memory subsets, accompanied by transcriptional programs indicative of exhaustion in relapse-associated marrow [ 163 ].

The immunosuppressive microenvironment amplifies these defects [ 164 ]. Regulatory T cells are expanded with enhanced suppressive capacity [ 165 ], and myeloid-derived suppressor cells (MDSCs) accumulate in both blood and marrow [ 8 , 164 ]. Leukemic progenitors maintain inhibitory signaling via PD-L1 and Galectin-9 expression, secretion of TGF-β and IL-10, and downregulation of MHC molecules [ 8 ]. Persistent exposure to these cues reduces cytokine production and disrupts the assembly of mature immunological synapses, thereby impairing target cell engagement [ 166 ]. Synapse formation defects are accompanied by incomplete recruitment of proximal signaling components, with reduced activities of Lck and ZAP70, leading to attenuation of downstream activation cascades [ 167 – 169 ]. In CAR-T cells, such proximal impairments limit proliferation and cytokine secretion despite intact CAR expression [ 167 ].

Metabolic dysregulation in AML further restricts immune cell function. Leukemic blasts rely on aerobic glycolysis, leading to intensive glucose consumption and release of inhibitory metabolites such as lactate [ 170 , 171 ]. In the setting of post-transplant relapse, the glycolytic capacity of T cells is substantially reduced [ 172 ]. PD-1 signaling suppresses glycolytic flux and, together with lactate accumulation and glucose depletion in the leukemic niche, limits the metabolic fitness of these cells [ 170 ].

These intrinsic and microenvironment-driven deficits are compounded by treatment-related injury. Induction regimens, including cytarabine with anthracyclines, exert profound lymphotoxicity [ 173 ]. Post-treatment T cells exhibit a reduced CD4/CD8 ratio, depletion of naïve (TN) cells with relative enrichment of effector memory (TEM) subsets, and persistent mitochondrial dysfunction that limits ex vivo expansion [ 173 , 174 ]. Such therapy-induced alterations aggravate exhaustion phenotypes present at diagnosis and further constrain manufacturing feasibility. In addition to these effects, the lymphodepleting chemotherapy preceding CAR-T infusion imposes further immunologic and hematopoietic stress. Standard conditioning with fludarabine and cyclophosphamide (Flu/Cy) depletes regulatory T cells and natural killer cells, thereby reducing competition for homeostatic cytokines [ 175 ]. This regimen also induces stromal and immune cells to secrete IL-7 and IL-15, which promote CAR-T cell proliferation and persistence [ 175 ]. In AML, baseline marrow aplasia and reduced hematopoietic reserve intensify the myelotoxic effects of lymphodepletion. Prolonged neutropenia exceeding 28 days and infection-related mortality rates above 20% have been reported in high-risk cohorts [ 1 ]. Excessive depletion extends cytopenia duration and increases susceptibility to infection, whereas insufficient depletion shortens CAR-T persistence and reduces anti-leukemic activity [ 175 ].

Leukemic blast contamination

In AML, contamination of leukapheresis products with myeloid blasts is common when circulating blasts are present and no upfront T-cell selection is performed [ 8 ]. In cohorts with hyperleukocytosis and clinical leukostasis, the peripheral blast percentage reaches a median of 90% (range 42–97%), indicating a substantial risk of blast co-collection during single-nuclear-cell apheresis [ 176 ]. Data from refractory or relapsed B-ALL demonstrate that the presence of circulating blasts results in a median leukemic-cell fraction of 32.7% in the starting apheresis material (range 3.23–83.2%) [ 177 ]. Such contamination is associated with reduced transduction efficiency (35.6% vs. 52.4%, p < 0.001) and impaired ex-vivo expansion (2.27-fold vs. 5-fold, p = 0.0055) [ 177 ]. In vivo, peak CAR-T proportion, absolute peak count, fold expansion, and persistence are all diminished, and non-response occurs more frequently (25% vs. 1.87%) [ 177 ]. Given the typically higher circulating blast burden observed in AML compared with B-ALL under hyperleukocytosis conditions, the true contamination level in AML is likely greater, which may compromise the quality of starting material and the performance of the final product.

Contamination introduces both direct and latent risks. Directly, reinfusion of unmodified leukemic blasts may reseed malignant cells [ 8 ]. Latently, blasts can be inadvertently transduced by viral vectors (lentivirus or retrovirus) during manufacturing, generating CAR-expressing leukemic cells (CAR-blasts) [ 178 ]. These cells may evade immune clearance through antigen masking, fratricide, or other mechanisms, and random vector integration can activate oncogenes or disrupt tumor suppressor genes, conferring proliferative or drug-resistant phenotypes [ 8 , 164 ]. Such events can promote treatment resistance or accelerate relapse. To mitigate these risks, manufacturing protocols often incorporate purification steps, such as magnetic bead sorting, to either positively select CD3⁺, CD4⁺, or CD8⁺ T cells or deplete myeloid marker–positive cells [ 8 ]. However, in AML, the limited T-cell yield makes extensive purification technically challenging, as it further reduces the available cell dose.

Blast contamination also impairs subsequent T-cell expansion. During culture, proliferating blasts compete with T cells for nutrients and cytokines, suppressing proliferation and lowering transduction efficiency. In B-ALL, the presence of circulating blasts significantly reduces final product expansion [ 177 ]. In AML, the higher blast proportion can drive a self-reinforcing cycle of contamination, intensified purification, T-cell loss, and expansion failure, ultimately hindering the ability to meet both quantitative and qualitative manufacturing requirements.

Timing of Re-infusion of CAR T cells

In AML, the timing of CAR-T cell infusion varies considerably and is typically categorized into the relapse or high disease burden phase and the remission or MRD-positive phase. Unlike lymphoid malignancies, patients with AML usually undergo multiple courses of intensive chemotherapy before re-infusion. This treatment history results in markedly reduced bone marrow reserve and necessitates additional lymphodepletion chemotherapy to facilitate CAR-T cell expansion. The combination of profound marrow aplasia and impaired immune competence substantially increases the risk of treatment-related toxicity.

In the high disease burden setting, abundant tumor-associated antigens provide persistent and robust stimulation to CAR-T cells, which promotes greater in vivo expansion and potent cytolytic activity [ 179 ]. In a phase II trial of CD19 CAR-T cells in t(8;21) AML, all ten treated patients achieved CR/CRi; however, grade ≥ 3 myelosuppression occurred in every case, and 53.3% experienced relapse within 12 months [ 180 ]. Similarly, in an enhanced CD33 CAR-T study ( NCT04835519 ), two of four relapsed AML patients achieved MRD-negative CRi, yet al.l developed grade II-IV cytopenia, with one patient experiencing grade 4 CRS. These findings suggest that high disease burden infusion may be an effective bridge to allo-HSCT in patients lacking alternative therapeutic options, although the substantial risk of severe toxicity must be carefully weighed.

Infusion during morphological remission in the presence of MRD aims to eradicate residual leukemic cells and thereby reduce relapse risk. Lower tumor burden in this context is associated with a reduced incidence and severity of CRS and comparatively milder myelosuppression. For instance, in the ongoing phase I/Ib PRGN-3006 trial ( NCT03927261 ) of a CD33 UltraCAR-T platform, no dose-limiting toxicities have been observed in the MRD-positive cohort, and the therapy has demonstrated a favorable safety profile along with measurable antileukemic activity in some patients. Nevertheless, this strategy encounters the challenge of antigen starvation, in which low antigen density may fail to sustain CAR-T cell proliferation and function, leading to limited durability of response [ 179 ]. The long-term clinical benefit of MRD-stage infusion remains uncertain and requires confirmation in larger, prospective trials.

Optimization strategies

Fig. 1 Targeting and recognition strategies in CAR-T therapy for AML. ( A ) Logic-Gated CAR-T Cells: Different logic-gated CAR designs improve AML targeting specificity and reduce toxicity. OR-gated CAR-T cells recognize either CD33 or CD123 to prevent antigen escape. AND-gated CAR-T cells require both antigens for activation, reducing off-leukemia toxicity. NOT-gated CAR-T cells incorporate an inhibitory receptor (iCAR) to prevent the targeting of normal hematopoietic stem cells (HSPCs). IF-BETTER CAR-T cells refine antigen recognition based on antigen density, while IF-THEN CAR-T cells use a SynNotch receptor to sequentially regulate CAR expression. ( B ) Nanobody-Based CAR-T Cells: CAR-T cells using single-domain antibodies (nanobodies, VHH) as the antigen-recognition domain. Their low molecular weight, stability, high affinity, and engineering flexibility reduce immunogenicity, prevent self-aggregation and self-activation, and delay T-cell exhaustion. ( C ) Adapter CAR-T Cells: Adapter-based CAR-T systems allow dynamic antigen targeting through soluble adapter molecules, enabling multi-targeting while reducing on-target off-leukemia toxicity. Removal of the adapter leads to CAR-T cell inactivation, acting as an intrinsic safety switch. Created with BioRender.com

Innovative Multi-targeting strategies: Logic-Gated CAR-T cells

OR-gated CAR constructs are activated by either target antigen, thereby broadening antigen coverage and reducing immune escape in AML, where antigen heterogeneity is prevalent (Fig. 1 A). In the CD33/CLL1 configuration, dual-targeted CAR-T cells exhibited potent cytotoxicity and repeated killing capacity against either antigen-positive AML cells in vitro, including HL-60, MV4-11, THP-1, U937, MOLM-13, and primary patient samples [ 181 ]. In an NSG xenograft model (U937-Luc), infusion of approximately 3 × 10⁶ CAR-T cells achieved durable tumor control, prolonged survival, and prevented relapse [ 181 ]. This strategy has progressed to early-phase clinical evaluation, as exemplified by NCT05016063 in AML/MDS. A similar OR-gate approach combining FLT3 and CD33 targets extended therapeutic coverage to both leukemic blasts and leukemic stem cells, resulting in prolonged survival in AML xenograft models [ 182 ]. The same antigenic logic has been implemented on an off-the-shelf CAR-NK platform (SENTI-202: CD33 OR FLT3 NOT EMCN), which demonstrated robust cytotoxicity against multiple primary AML samples in vitro and is currently under investigation in a Phase I trial ( NCT06325748 ).

The AND-gated CAR strategy requires concurrent expression of both target antigens for full activation, thereby minimizing on-target, non-leukemic toxicity (Fig. 1 A). Boucher et al. developed a CD33/CD123 split-signaling platform, distributing 4-1BB and CD3ζ domains between CARs specific for CD33 and CD123, respectively [ 183 ]. In vitro, cytotoxicity and IL-6 release were observed only against dual-antigen targets, including engineered CHO-CD33/CD123 cells and the AML cell line MOLM-13 [ 183 ]. In vivo, tumor suppression in AML NSG xenografts matched that of single-target CARs, while CD34⁺ hematopoietic cells remained unaffected in humanized mice [ 183 ]. PLCγ1, substituted for conventional CD3ζ, produced a more stringent AND gate, activated exclusively in the presence of both antigens in Nalm6 (CD19/HER2/ROR1 variant), CHLA-255 (neuroblastoma), and 143B (osteosarcoma) models, eliminating tumors in a ROR1 pneumotoxicity model without lethality [ 184 ].

The IF-BETTER gating approach maintains effector function under low antigen density by overlaying a costimulatory receptor (CCR) onto the primary CAR, thereby modulating activation thresholds in an antigen-density–dependent manner (Fig. 1 A). Haubner et al. (ADCLEC.syn1) combined an ADGRE2-CAR with a CLEC12A-CCR in a MOLM-13 model [ 140 ]. The CCR augmented cytotoxicity and IFN-γ/IL-2 release when ADGRE2 expression is low, prolonged survival at reduced CAR-T doses in NSG AML xenografts, and preserved CD34⁺ hematopoietic progenitors in r/r AML PDX and HSPC-AML co-engraftment models; a phase I trial is ongoing ( NCT05748197 ) [ 140 ]. Katsarou et al. paired a CD33-CAR with a CLEC12A-CCR, enhancing tumor clearance and cytokine release in low-antigen-density K562-derived AML lines and the Nalm6 model, while reducing exhaustion markers such as PD-1 and TIM-3 [ 185 ]. In NSG xenografts, this dual-receptor configuration surpassed single CAR constructs in eliminating low-antigen tumors [ 185 ].

NOT-gated CAR designs incorporate inhibitory signaling to prevent on-target, non-leukemic toxicity (Fig. 1 A). Fei et al. generated an HLA-DR–directed inhibitory CAR (iCAR) and co-expressed it with CD33- or CD19-specific CARs [ 186 ]. In MOLM-13, THP-1, HEL cells and their HLA-DR–high/low variants, as well as patient-derived AML progenitor cells, the iCAR suppressed CAR-T activation in an HLA-DR–dependent manner [ 186 ]. DR-overexpressing CD34⁺ hematopoietic stem/progenitor cells and monocytes inhibited cytokine release and cytotoxicity, whereas HLA-DR^low/neg leukemia cells were efficiently eliminated [ 186 ]. The inhibitory effect correlated with HLA-DR expression levels [ 186 ]. In NSG MOLM-13-luc xenografts, iCAR-modified CAR-T cells maintained anti-leukemic efficacy, prolonged survival, and reduced myelosuppression in a humanized CD34⁺ HSC model [ 186 ]. Building on this principle, Frankel et al. integrated a FLT3/CD33 OR-activation module with an endomucin (EMCN)-specific iCAR in a CAR-NK platform [ 182 ]. EMCN is a surface glycoprotein highly expressed on normal HSCs but absent on AML blasts and LSCs, serving as a protective marker that helps preserve healthy hematopoiesis [ 182 ]. The EMCN-iCAR suppressed NK activation in the presence of EMCN⁺ hematopoietic cells while preserving tumor clearance in an NSG AML model [ 182 ]. This OR-NOT configuration (SENTI-202) advances to Phase I testing ( NCT06325748 ) to evaluate dosing, safety, and early efficacy.

IF-THEN–gated CARs employ SynNotch receptors to induce CAR expression only after recognition of a predefined gating antigen, introducing a temporal delay before T-cell activation (Fig. 1 A). Choe et al. used mesothelin, EphA2, or HER2 as gating antigens and IL13Rα2 or HER2 as killing antigens [ 187 ]. CAR expression and cytotoxicity occurred exclusively in dual-antigen contexts, as shown in U87, A549, and K562-derived models, thereby reducing cytokine release under single-antigen conditions [ 187 ]. In NSG xenografts and multifocal tumor models, this design enabled selective clearance of dual-antigen foci, prolonged tumor control, and mitigated T-cell exhaustion [ 187 ]. In AML, however, the co-localization of leukemic and normal hematopoietic cells within the bone marrow niche may limit spatial discrimination, potentially affecting clinical feasibility.

Nanobody-based CAR-T cells: structural innovation for enhanced targeting

Nanobodies are applied in CAR-T antigen recognition domain design because of their low molecular weight, structural stability, high affinity, and suitability for humanization and engineering [ 188 ] (Fig. 1 B). Compared with conventional scFvs, they reduce immunogenicity, limit self-aggregation and construct self-activation, and delay T-cell exhaustion [ 189 ]. Although clinical use in AML is still emerging, preclinical and early clinical studies across multiple antigens confirmed feasibility and proposed structural and therapeutic refinements, establishing a basis for improving efficacy and safety.

In AML, CD7 is the only antigen for which nanoantibody-based CAR-T therapy has reached clinical evaluation. Approximately 30% of AML bone marrow-derived primary cells express CD7. Zhang et al. developed a naturally selected CD7 CAR-T with a dual-VHH (dVHH) architecture (NS7 CAR-T) and tested it in a phase I trial ( NCT04938115 ) enrolling 10 patients with R/R CD7⁺ AML [ 190 ]. Complete remission occurred in 70% of cases, with several achieving long-term disease-free survival after a second hematopoietic stem cell transplantation. Relapse was primarily linked to CD7 loss [ 190 ]. Treatment was well tolerated, with mild cytokine release syndrome in 80% of patients and no neurotoxicity [ 190 ].

Cheng et al. created two CD70 CAR-T constructs with distinct VHH domains [ 65 ]. Both induced selective cytotoxicity against AML cell lines and patient-derived samples and delayed tumor progression in NSG mice. However, CD70 expression varied markedly among patients, with only 40.4% exceeding the therapeutic threshold [ 65 ]. To address antigen heterogeneity, Zhang et al. designed a CD13/TIM3 bispecific nanoantibody CAR-T targeting AML cells and leukemia stem/progenitor cells [ 191 ]. This construct produced potent, selective cytotoxicity in multiple AML models and achieved sustained tumor eradication in vivo without detectable toxicity [ 191 ].

Lessons from other hematologic malignancies inform AML optimization strategies. For CD72 CAR-T cells, moderate affinity enhancement or framework modification improves cytotoxicity in low-antigen models, whereas excessive affinity offers no additional benefit. Humanization reduces immunogenicity without impairing function [ 192 ]. Bispecific approaches in non-AML contexts (e.g., CD19/CD20, CD30/CD5) demonstrate the value of multi-antigen targeting in relapse prevention [ 193 , 194 ]. Data from solid tumors also provide cautionary guidance. B7-H3 nanoantibody CAR-T cells, while active against glioblastoma, induced on-target, off-tumor toxicity due to cross-reactivity with low-level B7-H3, emphasizing the need to balance potency and safety in AML target selection and antigen sensitivity tuning [ 195 ].

Adapter CAR-T cells: enhancing safety and translational potential

Adapter CAR-T (AdCAR-T) technology represents a modular strategy designed to enhance antigen specificity, therapeutic control, and safety in AML treatment [ 196 ] (Fig. 1 C). Unlike conventional CAR-T therapies with fixed antigen specificity, AdCAR-T platforms employ soluble adapter molecules to dynamically regulate antigen recognition, bridging CAR-T cells to AML-associated targets [ 197 ]. This modularity enables sequential and simultaneous multi-targeting, addressing antigen escape while minimizing off-leukemia toxicity [ 197 ].

In vitro studies by Atar et al. demonstrated that AdCAR-T cells directed against CD33, CD38, and CD371 exerted enhanced cytotoxicity in U937 derivatives and primary AML blasts [ 198 ]. In NSG mice bearing either U937 xenografts or pediatric AML PDX, multi-adapter treatment delayed or prevented relapse and achieved more durable tumor clearance and survival benefit compared with single-adapter therapy [ 198 ]. Volta et al. reported that in MOLM14 cells and primary AML samples, a CD33 + CD117 dual-adapter approach increased adapter density on the tumor surface and augmented cytotoxicity [ 199 ]. In corresponding NSG xenograft models, the dual-adapter group achieved faster and more sustained leukemia eradication than the single-target group [ 199 ].

The modular concept is further exemplified by bridging protein CAR-T technology. Rennert et al. engineered CAR19 T cells to secrete biparatopic bridging proteins capable of redirecting recognition to CLEC12A or to CLEC12A + CD33 [ 200 ]. This platform showed superior tumor suppression and extended survival compared with direct CLEC12A CAR-T cells in AML cell lines including U937, MOLM14, and PL-21, as well as in NSG xenograft models [ 200 ]. These findings underscored the potential of multiple antigens targeting to counteract immune escape.

The transient binding of both adapter and bridging protein platforms functions as an intrinsic safety switch. Withdrawal of the adapter or cessation of bridging protein secretion rapidly terminates CAR-T activity, reducing the risk of myelosuppression arising from target expression on normal hematopoietic progenitors. Clinically, UniCAR-T-CD123 has reversed hematologic toxicity in relapsed or refractory AML patients upon discontinuation of the adapter [ 16 ], and a phase I clinical trial ( NCT04230265 ) is currently evaluating its safety and preliminary efficacy.

Despite these advantages, optimization of AdCAR-T for AML remains challenging. Continuous adapter infusion requires precise pharmacokinetic control to maintain therapeutic engagement while minimizing adverse effects [ 197 ]. Current AdCAR-T platforms often require higher T-cell doses to achieve cytotoxicity comparable to conventional CAR-T cells [ 16 ], and the short half-life of adapter molecules necessitates carefully designed dosing schedules to ensure sustained antitumor activity [ 199 , 201 ].

Fig. 2 Functional and persistence-enhancing strategies in CAR-T therapy for AML. ( A ) Epigenetic & Pharmacologic Modulation: Hypomethylating agents (HMA), histone deacetylase inhibitors (HDACi), and all-trans retinoic acid (ATRA) enhance target antigen expression (e.g., CD70, CD123, CD38), improving AML recognition and CAR-T cytotoxicity. ( B ) Gene Editing: Epitope editing or gene knockout strategies (e.g., CD33-KO in HSPCs) prevent CAR-T-mediated myelotoxicity while preserving normal hematopoiesis, allowing selective AML eradication. ( C ) Metabolic & Epigenetic Reprogramming: Metabolic modulation (e.g., mTOR inhibition with Rapamycin) enhances memory-like CAR-T phenotypes, improving persistence. Bcl-2 inhibition reduces activation-induced cell death (AICD). Epigenetic interventions (HMA/HDACi) and PD-1 knockout prevent CAR-T exhaustion and enhance cytotoxicity. Created with BioRender.com

Epigenetic and pharmacologic strategies to boost antigen expression for CAR-T targeting

Epigenetic modulation is recognized as an effective approach to enhance antigen expression in AML cells (Fig. 2 A). Treatment with the demethylating agents decitabine (DAC) and azacitidine (AZA) increased the surface density of CD70 and CD123, thereby improving the susceptibility of AML cells to targeted CAR-T cytotoxicity [ 65 , 202 – 204 ]. In THP-1 cells and primary AML samples, short-term pretreatment of CD123 CAR-T cells with DAC (0.25–1.0 µM for 48 h) significantly augmented their in vitro cytotoxicity [ 202 ]. In a THP-1-luciferase NSG mouse model, this approach achieved faster and more complete leukemia clearance compared with either treatment alone [ 202 ].

AZA produced similar effects without elevating CD123 expression on healthy hematopoietic cells. In KG1a, MOLM-13, and multiple primary AML samples, AZA increased CD123 expression and sensitized cells to CAR-T killing [ 205 ]. In MOLM-13-luc and OCI-AML3 NSG xenografts, an “AZA followed by CD123 CAR-T” regimen prolonged tumor-free survival relative to single-agent therapy, accompanied by higher CD4⁺ CAR-T activation marker expression and increased cytokine production [ 205 ].

Beyond DNA methylation inhibitors, small-molecule inducers have been used to improve antigen accessibility. All-trans retinoic acid (ATRA, 10 nM for 18 h) rapidly and reversibly increased CD38 expression in HL-60, U937, KG-1, and patient-derived AML blasts, thereby enhancing the cytotoxic activity of CD38 CAR-T cells in vitro [ 34 ]. In addition, ATRA induced the expression of NKG2D ligands, including MICA/B and members of the ULBP family, in AML cell lines such as HL-60 and U937 and in primary AML samples, thereby increasing their susceptibility to NKG2D-mediated immune cytotoxicity [ 206 ].

Gene editing for AML: bridging precision engineering and clinical application

Gene editing of HSPCs can either modify antigen epitopes with nucleotide-level precision or completely delete the antigen (Fig. 2 B). Both approaches block recognition by CAR-T cells or therapeutic antibodies while preserving hematopoietic function. Protecting edited HSPCs from on-target cytotoxicity enables selective clearance of unmodified AML cells and minimizes prolonged myelosuppression.

Epitope editing has been used to introduce single-nucleotide substitutions at critical residues to abolish CAR or antibody binding without impairing the protein’s physiological role. In human CD34⁺ HSPCs, CRISPR–Cas9 combined with a single-stranded oligonucleotide donor introduced a defined point mutation within the extracellular domain of CD123 [ 207 ]. This modification preserved IL-3 receptor signaling yet eliminated recognition by CD123 CAR-T cells and monoclonal antibodies [ 207 ]. Edited progeny demonstrated resistance to CAR-T-mediated cytolysis in vitro, whereas unmodified AML cell lines (MOLM-13, KG-1) remained fully susceptible [ 207 ]. In NSG mice, transplantation of edited HSPCs supported durable multilineage hematopoiesis for at least sixteen weeks in the presence of CD123 CAR-T infusion [ 207 ]. Adenine base editing of the CD45 epitope yielded a site-specific mutation that disrupted binding to affinity-optimized CD45-CARs while retaining the physiological functions of CD45 [ 208 ]. Consistent with this approach, Garaudé et al. demonstrated that precise epitope editing of CD45 could selectively abolish CAR recognition (e.g., BC8 or MIRG451 epitopes) without impairing CD45 phosphatase function, thereby preserving normal hematopoiesis during CD45-directed therapy [ 209 ]. In NBSGW mice, edited HSPCs maintained multilineage chimerism for twenty weeks despite repeated CAR-T administrations [ 208 ]. Similarly, Casirati et al. applied base editing to introduce precise amino-acid substitutions in FLT3, CD123, and KIT, thereby abolishing CAR or antibody recognition while preserving receptor function and hematopoietic competence [ 210 ].

Whereas epitope editing preserves antigen function, CRISPR-Cas9-mediated knockout abolishes immune recognition by removing the antigen entirely. Kim et al. demonstrated that CRISPR/Cas9-mediated CD33 knockout in human HSPCs generated CD33⁻ myeloid cells resistant to anti-CD33 CAR-T cytotoxicity while maintaining normal hematopoietic differentiation in vivo [ 211 ]. Gurney et al. targeted coding exons of CD38 in cord blood– or peripheral blood–derived CD34⁺ HSPCs, achieving efficient deletion. In vitro, CD38-null HSPCs differentiated into myeloid and lymphoid lineages without impairment of hematopoietic potential [ 36 ]. In NSG mice, CD38-knockout HSPC transplantation sustained multilineage hematopoietic reconstitution for at least twenty weeks under repeated exposure to affinity-optimized CD38 CAR-NK [ 36 ]. Co-transplanted CD38⁺ leukemia cells were selectively eradicated [ 36 ]. Although knockout strategies prevent same-target hematopoietic toxicity, they require careful selection of nonessential antigens and long-term functional monitoring in preclinical models.

Metabolic and epigenetic strategies to enhance CAR-T cell persistence in AML

CAR-T persistence in AML is limited by progressive T-cell depletion and accelerated differentiation. Metabolic reprogramming helps maintain a less differentiated phenotype and delays functional decline through modulation of key signaling pathways. Targeting the PI3K/AKT/mTOR axis enhances CAR-T expansion and preserves memory-like phenotypes (Fig. 2 C). In NOD/SCID xenograft models, inhibition of mTOR with rapamycin significantly increased in vivo CAR-T persistence and augmented cytotoxicity [ 212 ]. Notably, rapamycin-treated CAR-T cells maintained over 70% viability at day 14 compared with less than 40% in controls and achieved a two-fold increase in specific lysis of AML targets [ 212 ]. Inhibitors of Bcl-2, such as venetoclax, prolonged CAR-T survival by reducing activation-induced cell death without diminishing cytolytic activity [ 212 ].

Beyond metabolic modulation, immune checkpoint regulation also shapes CAR-T persistence. Elevated PD-1 expression is closely associated with T-cell exhaustion. In NSG mice engrafted with CLEC12A⁺ AML, PD-1 gene silencing in CLEC12A CAR-T cells prolongs survival and maintains durable anti-leukemic activity [ 213 ]. In these models, PD-1–deficient CAR-T cells expand 2.3-fold more in vivo and extend median survival from 28 to 54 days, highlighting PD-1 blockade as an effective adjunct to enhance CAR-T durability [ 213 ].

Epigenetic reprogramming provides an additional means to reinforce CAR-T function in AML (Fig. 2 C). Treatment of AML cells with the hypomethylating agents DAC or AZA mitigated T-cell exhaustion by downregulating inhibitory markers [ 214 ]. In NSG xenograft models established with THP-1-luc or MOLM-13-luc cells, DAC pretreatment followed by CD44v6 CAR-T infusion significantly improves in vivo CAR-T expansion and persistence, resulting in sustained leukemia clearance and extended overall survival [ 214 ]. This sequential regimen reduces leukemic burden by more than 90% within two weeks and maintains remission for over 60 days [ 214 ].

Fig. 3 Allogeneic CAR-T strategies for AML. Gene-edited allogeneic CAR-T cells prevent graft-versus-host disease primarily through TCR knockout, while HLA/MHC knockdown and CD52 knockout (in combination with anti-CD52 antibody–mediated lymphodepletion) mitigate host-mediated allogeneic rejection, thereby enhancing CAR-T persistence and AML clearance. Created with BioRender.com

Expanding CAR-T accessibility: the promise of allogeneic approaches

Allogeneic CAR-T (Allo-CAR-T) cells are manufactured in bulk from healthy donors, shortening production timelines and lowering costs. In rapidly progressing hematologic malignancies such as AML, these cells demonstrated high persistence and potent cytotoxicity in vivo because they remained unaffected by chemotherapy. The primary barriers to clinical translation are GVHD and host immune rejection. GVHD results from recognition of host tissues by donor TCRs, whereas immune rejection arises from rapid clearance driven by HLA mismatch (Fig. 3 ).

To mitigate GVHD, Tipanee et al. applied the Sleeping Beauty transposon system to introduce CD19-28ζ CARs and used CRISPR/Cas9-mediated TRAC disruption to generate TCR-negative CAR-T cells [ 215 ]. In a NALM6-luciferase B-ALL mouse model, a single infusion achieved complete tumor eradication at both high and low tumor burdens. In vivo imaging confirmed durable remission without significant GVHD [ 215 ].

Wang et al. reduced MHC expression by introducing Kaposi’s sarcoma-associated herpesvirus (HHV-8)-derived ubiquitin ligases K3 or K5 into CAR-T cells. K3 downregulated MHC-I (HLA-A/B/C) and MHC-II (HLA-DR) and enhanced in vivo expansion in an NSG mouse allogeneic T-cell model [ 216 ]. K5 decreased NK cell-mediated killing of MIC-A/B–positive K562 target cells, indicating reduced innate immune clearance [ 216 ]. To evade both T- and NK-cell-mediated rejection, Degagné et al. engineered BCMA CAR-T cells (CB-011) using a CRISPR-based gene-editing approach [ 217 ]. The design included BCMA CAR knock-in at the human TRAC locus, B2M knockout, and insertion of a B2M-HLA-E fusion protein [ 217 ]. These cells mediated BCMA-specific cytotoxicity in myeloma cell lines (MM.1 S, NCI-H929) and xenograft models, while remaining undetected by allogeneic T cells and NK cells [ 217 ]. CB-011 is currently under evaluation in a phase I clinical trial for R/R multiple myeloma (CaMMouflage, NCT05722418 ).

Porreca et al. developed an RNA aptamer-guided “Pin-point” base editing platform capable of simultaneously knocking out B2M, TRAC, CD52, and PDCD1 while inserting CAR transgenes at defined genomic sites without creating DNA double-strand breaks [ 218 ]. This approach achieved high editing efficiency in primary T cells without detectable chromosomal translocations [ 218 ]. The resulting CAR-T cells resisted both adaptive and innate immune clearance in vitro and showed delayed functional exhaustion during prolonged antigen exposure [ 218 ].

For safety control, Sommer et al. designed an FLT3-targeted Allo-CAR-T for AML with an in-cis rituximab-responsive elimination switch [ 219 ]. In an EOL-1-luciferase AML xenograft model, the CAR-T cells rapidly cleared leukemia but also attacked normal hematopoietic progenitors [ 219 ]. Administration of rituximab during tumor clearance selectively depleted the CAR-T population, facilitating bone marrow recovery and mitigating hematopoietic toxicity associated with shared antigen expression [ 219 ].

Sequential CAR-T and HSCT

The integration of CAR-T therapy with hematopoietic stem cell transplantation (HSCT) is investigated as a sequential approach to improve outcomes in R/R AML. In bridging settings, CAR-T infusion is administered to achieve remission and reduce leukemic burden before HSCT [ 220 ]. CD33 CAR-T induced CR in 41.7% of patients relapsing after HSCT (5 of 12), with predominantly grade 1–2 CRS and no ICANS; several responders proceeded to a second transplant, whereas one death from sepsis occurred during prolonged neutropenia [ 221 ]. CD7 CAR-T produced CR in 7 of 10 patients, yet four of these remissions relapsed within 100 days, indicating that sustained disease control is typically achieved only after subsequent allogeneic HSCT [ 222 ]. Donor-derived CLL-1 CAR-T administered on day 11 of remission resulted in prompt leukocyte, chimerism, and platelet engraftment without GVHD at four months, demonstrating feasibility of rapid bridging [ 29 ]. Post-CAR-T immune activation is considered a potential barrier to post-transplant immune reconstitution. However, current observations are limited to sporadic liver enzyme elevations variably attributed to CRS or possible GVHD, with no consistent evidence of increased GVHD incidence or engraftment failure [ 220 ]. The efficacy of post-transplant CAR-T maintenance and the optimal infusion window relative to immunosuppression have not been defined, underscoring the need for prospective trials to establish sequencing and dosing strategies.

Conclusion

The AML antigen repertoire is largely confined to preclinical candidates, emphasizing the need for therapeutic designs that reconcile specificity with safety. Translation into clinical practice is hindered by antigen overlap with normal progenitors, heterogeneous and dynamic expression, high leukemic burden within immunosuppressive marrow niches, and manufacturing limitations such as narrow collection windows and impaired T-cell quality. Current development focuses on aligning CAR-T platform design with this antigen landscape to enhance efficacy while limiting toxicity. Logic-gated and adapter CAR constructs broaden recognition and reduce myelotoxicity, nanobody-based CARs provide stable low-immunogenic binding, and gene-edited hematopoietic stem and progenitor cells enable selective AML clearance without prolonged marrow suppression. Metabolic and epigenetic modulation sustain T-cell persistence in hostile niches, while allogeneic CAR-T platforms can bypass autologous manufacturing constraints. In selected settings, integration with hematopoietic stem cell transplantation consolidates remission and restores hematopoiesis. Next-generation CAR-T therapies are therefore expected to play defined roles within the AML treatment continuum—serving as bridging or consolidation strategies for high-risk and refractory disease, and potentially extending to frontline settings as antigen-restricted and universal platforms mature. However, durable in vivo persistence without myelotoxicity, precise control of cytokine-driven toxicity, and scalable manufacturing remain major challenges that will determine the feasibility of broad clinical translation. Collectively, these advances outline a realistic pathway toward integrating CAR-T therapy as a safe, durable, and potentially transformative modality in AML management.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xumeng Zhao and Xi Ming are contributed equally to this work and share first authorship.

Author contributions

XMZ and XM contributed equally to this work and are co–first authors. They jointly conceived the idea for the article, developed the outline, and contributed to critical discussions throughout the manuscript preparation. JYW performed the literature search, analyzed the referenced data, drafted the initial version of the manuscript, and prepared all figures. XJZ and YX supervised the project and provided critical revisions for important intellectual content. All authors reviewed and approved the final version of the manuscript.

Funding

This review was supported by the National Natural Science Foundation of China (No. 81873444, No. 82070213, and No.82370196).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval

Not applicable.

Informed consent

Not applicable.

Animal welfare statement

Not applicable.

Competing interests

The authors declare no competing interests.

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