Challenges and advances in CAR-T cell therapy for B-ALL

Abstract

Chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment landscape for hematological malignancies such as relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), with several CAR-T products now approved globally for R/R B-ALL. Despite high initial response rates, major challenges remain, including disease relapse due to antigen escape and the limited persistence of CAR-T cells; treatment-related adverse events such as cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, hematologic toxicity, and infections; and limited access to CAR-T therapy. In this review, we discuss the limitations of CAR-T and strategies to overcome them, specifically in the context of B-ALL, including the use of allogeneic CAR-T, dual-targeted CAR-T, and combination strategies with novel technologies and agents. Furthermore, we explored the role of consolidative allogeneic hematopoietic stem cell transplantation after CAR-T therapy and the potential of integrating CAR-T into the first-line treatment for B-ALL. Future research should aim to increase the efficacy of CAR-T, reduce their toxicity, improve their accessibility, and expand their use to earlier lines of therapy for B-ALL.

Background

The advent of chimeric antigen receptor T-cell (CAR-T) therapy has significantly improved the prognosis of patients with relapsed/refractory (R/R) B-ALL (Table 1). As of April 2026, three CAR-T products have been approved in the United States for the treatment of R/R B-ALL: Tisagenlecleucel (tisa-cel, 2017), Brexucabtagene Autoleucel (brexu-cel, 2021), and Obecabtagene Autoleucel (obe-cel, 2024). In their respective clinical trials, these three CAR-T products achieved complete remission (CR) rates ranging from 71% to 81%, demonstrating favorable long-term responses [1–3]. In China, Inaticabtagene Autoleucel (inati-cel, 2023), which was developed by our institution, became the first CAR-T product approved for the treatment of adult R/R B-ALL in China. In a pivotal clinical trial, Inati-cel achieved a CR rate of 85.4%, with a 2-year relapse-free survival (RFS) rate of 35.8% and a 2-year overall survival (OS) rate of 55.2% [4]. Furthermore, a growing number of clinical trials of CAR-T therapy for R/R B-ALL are emerging.

Table 1.

Summary of commercial CAR-T for R/R B-ALL worldwide and their pivotal trial results

Drug name	Year of approval	First approval country	Manufacturer	scFv	Costimulatory domain	Trial ID/Reference	Patient number	Median age	Dose	CR/CRi rate	CRS/sCRS	ICANS/sICANS (neurotoxicity)	Long term outcome
Tisagenlecleucel	2017	US	Novartis	CD19/ FMC63	4-1BB	NCT02435849 [1]	75	11 (Range: 3–23)	0.2–5.4*10^6/kg	81% (61/75, 61 MRD negative)	58/35	30/10	6-month EFS: 73%; 12-month EFS: 50%; 6-month OS: 90%; 12-month OS: 76%; 6-month RFS: 80%; 12-month RFS: 59%
Brexucabtagene autoleucel	2021	US	Kite Pharma	CD19/ FMC63	CD28	NCT02614066 [2]	55	40 (IQR: 28–52)	10^6/kg	71% (39/55, 38 MRD negative)	49/13	33/14	Median DOR: 12.8 months; Median RFS: 11.6 months; Median OS: 18.2 months
Obecabtagene Autoleucel	2024	US	Autolus Therapeutics	CD19/CAT	4-1BB	NCT04404660 [3]	127	47 (Range: 20–81)	BM disease-adjusted and split-dose schedule: day 1: 100*10^6, day 10: 310*10^6(≤ 20% blasts); day 1: 10*10^6, day 10: 400*10^6(>20% blasts)	78% (99/127, 85 MRD negative)	87/3	29/9	Median EFS: 11.9 months; 6-month EFS: 65.4%; 12-month EFS: 49.5%; Median OS: 15.6 months; 6-month OS: 80.3%; 12-month OS: 61.1%; Median DOR: 21.2 months
Inaticabtagene autoleucel	2023	China	Juventas Cell Therapy Ltd	CD19/HI19α	4-1BB	NCT04684147 [4]	48	32 (Range: 18–58)	0.4–0.6*10^8 CAR-T cells	85.4% (41/48, 41 MRD negative)	42/6	4/3	Median RFS: 12.4 months; 1-year RFS: 54.5%; 2-year RFS: 35.8%; 1-year OS: 72.1%; 2-year OS: 55.2%
Talicabtagene autoleucel	2023	India	Indian Institute of Technology, Bombay and Immunoadoptive Cell Therapy	CD19/humanized scFv	4-1BB	CTRI/2021/04/032727, CTRI/2022/12/048211 [5]	15	22 (IQR: 17–39)	≥ 5*10^6/kg	73% (11/15, 10 MRD negative)	13/3	0/0	Median PFS: 6 months; 12-month remission rate in CR patients: 5/9 (56%)
Puzolcabtagene autoleucel	2025	China	Chongqing Precision Biotech	CD19/humanized scFv	NA	NCT05334823[6]	64	11(Range: 3–21)	NA	90.6% (58/64, 57 MRD negative)	63/19	32/25	Median DOR: 10.61 months; Median OS: 23.92 months

BM: Bone marrow; CR: Complete remission; CRi: CR with incomplete hematologic recovery; CRS: Cytokine release syndrome; DOR: Duration of remission; EFS: Event free survival; ICANS: Immune effector cell associated neurotoxicity syndrome; IQR: Interquartile range; MRD: Minimal residual disease; PFS: Progression free survival; OS: Overall survival; RFS: Relapse free survival; scFv: Single-chain variable fragments; sCRS: Severe cytokine release syndrome; sICANS: Severe immune effector cell associated neurotoxicity syndrome

Despite these high initial response rates, major challenges remain, including treatment failure due to antigen escape and limited CAR-T persistence, treatment-related toxicity, and limited accessibility. In this review, we discuss these challenges and recent research progress aimed at overcoming them in the context of R/R B-ALL.

Challenges of CAR-T cell therapy in R/R B-ALL

Antigen loss

Antigen loss primarily arises from intrinsic mechanisms within tumor cells (Fig. 1); this can lead to CD19-negative relapse, which is a major cause of treatment failure and poor long-term survival in R/R B-ALL patients after CD19 CAR-T therapy. B-ALL cells can downregulate or lose CD19 antigen expression through various mechanisms: At the DNA level, acquired frameshift mutations in exons 2–5 of CD19 can result in truncated CD19 proteins lacking membrane anchorage, rendering them undetectable on the cell surface by both CD19 CAR-T cells and flow cytometry [7]. Hypermethylation of the CD19 gene promoter can also reduce CD19 expression, preventing recognition by CD19 CAR-T cells [8]. At the RNA level, alternative splicing of CD19 mRNA in B-ALL generates isoforms that cannot encode functional CD19 proteins, impairing CD19 CAR-T recognition and contributing to relapse [9, 10]. The RNA-binding protein YTHDF2 can downregulate CD19 expression via m6A-dependent mRNA degradation, contributing to antigen escape from CD19 CAR-T therapy [11]. Dysregulated glycosylation of CD19 at the posttranslational level may also affect CD19 CAR-T recognition by B-ALL cells [12]. Lymphoid-to-myeloid lineage switching, which is particularly common in KMT2A-rearranged ALL, represents another mechanism of antigen-negative relapse driven by lineage plasticity under CAR-T immune pressure [13, 14]. In addition, preexisting subclones of CD19-negative leukemic blasts have been identified in some ALL patients prior to CAR-T therapy. Under selective pressure from CAR-T cells, these negative subclones may expand and eventually relapse [15, 16]. Furthermore, inadvertent transduction of the CD19 CAR transgene into B-ALL blasts during CAR-T manufacture and subsequent reinfusion into the patient can also cause CD19-negative relapse. This occurs due to cis-binding and masking of the CD19 epitope by the CAR expressed on the blast surface, preventing recognition by both CAR-T cells and flow cytometry [17].

Fig. 1.

Overview of CD19 escape mechanisms in B-ALL. Figure created with Biorender

CAR-T-cell recognition and activation depend on the high-density expression of target antigens. Therefore, the downregulation of antigens on B-ALL blasts may enable immune escape [18]. Previous CD19-targeted therapies (e.g., blinatumomab) can cause CD19 downregulation [19–22]. Additionally, studies have shown that the interaction between B-ALL cells and CD19 CAR-T cells induces CAR-mediated internalization and degradation of CD19 on B-ALL cells [23, 24]. Moreover, trogocytosis by CD19 CAR-T cells can transfer CD19 from B-ALL cells to themselves, reducing the CD19 density on B-ALL cells and impairing CAR-T-mediated death [25]. Interestingly, CD19 monoclonal antibodies can promote the rapid dissociation of CD19 CAR-T cells from B-ALL cells, mitigating trogocytosis-induced CD19 loss. The combination of CD19 CAR-T and anti-CD19 mAbs has synergistic anti–B-ALL effects [26], but there is limited clinical evidence supporting this combination strategy.

Limited CAR-T function and persistence

CAR-T intrinsic factors, such as impaired function, inadequate expansion, poor persistence, and exhaustion, also present significant barriers to treatment efficacy and can lead to refractory disease or antigen-positive relapse. From manufacturing to infusion, each step may influence in vivo activity, and the tumor microenvironment can further exacerbate these limitations.

The differentiation stage of the T cells used in manufacturing significantly influences CAR-T-cell persistence. CAR-T cells derived from early differentiation stages (naive T cells, central memory T cells, and stem cell-like memory T cells) exhibit superior expansion and persistence and are correlated with favorable outcomes [27, 28]. In contrast, terminally differentiated T cells are associated with poor expansion and relapse [29, 30]. Strategies to enrich early-differentiated T cells include subset selection, cytokine addition (IL-7, IL-15, and IL-21), CAR structural modification, and pharmacological or genetic regulation [31]. In a study by Aldoss et al., [32] CAR-T with naive/memory T-cell–selection achieved an 87% CR/complete remission with incomplete hematologic recovery (CRi) rates, with 1-year RFS and OS rates of 52.6% and 62.3%, respectively. Further clinical studies are needed to determine whether these enrichment strategies for early differentiation T cells can provide additional benefits to R/R B-ALL patients.

CAR design also significantly affects in vivo activity. Moderately reducing single-chain variable fragment (scFv) affinity may mitigate adverse effects without compromising antitumor function. Moreover, the characteristics of low-affinity scFvs, such as serial activation and rapid dissociation from the target antigen, may enhance CAR-T proliferation signals and in vivo persistence [33, 34]. Compared with that in tisa-cel and brexu-cel, the scFv in obe-cel has lower affinity (both derived from FMC63), which may partially explain the lower toxicity and improved persistence of obe-cel [3, 33]. Another factor is the immunogenicity of the scFv. Murine-derived scFvs can induce host anti-CAR immune responses, limiting their function and persistence [35]. Using humanized scFvs is a potential strategy to address this [36], but whether humanized CAR-T products are superior to murine-based products for treating R/R B-ALL remains uncertain.

CD28 and 4-1BB are the most commonly used costimulatory molecules. CD28 induces rapid activation and early activity, whereas 4-1BB has been shown to confer superior persistence in preclinical studies [37, 38]. In a head-to-head comparison, all patients in the 4-1BB group achieved minimal residual disease (MRD)-negative remission versus 89% in the CD28 group, with higher 1-year RFS and OS for 4-1BB [39]. However, conflicting reports exist: Some studies have reported no significant difference in long-term survival between the two [40, 41], and a real-world study reported comparable outcomes between tisa-cel (4-1BB) and brexu-cel (CD28) [42]. Given that multiple factors can influence CAR-T efficacy, larger randomized controlled trials are needed to analyze the effect of costimulatory domain selection on CAR-T therapy for R/R B-ALL.

After infusion, CAR-T cells rely on tumor antigen stimulation to activate their antitumor effector functions. However, persistent antigen exposure can drive CAR-T- exhaustion, limiting their activity and persistence and leading to relapse [43]. Numerous studies have confirmed that CAR-T cells post infusion exhibit exhaustion-associated transcriptional signatures and phenotypes, including the activation of exhaustion-related transcription factors and upregulation of immune checkpoints [44–46]. Additionally, antigen exposure initiates epigenetic reprogramming in CAR-T cells. Through longitudinal analysis of the methylation landscape in CD8 + CAR-T cells from R/R B-ALL patients, Zebley et al. [47] reported that effector genes in CD8 + CAR-T cells acquired demethylation within the first week post infusion, while memory-related genes gradually underwent methylation silencing. Concurrently, CAR-T cells transitioned toward an exhausted phenotype, as evidenced by demethylation at loci such as CX3CR1, BATF, and TOX, ultimately limiting their persistence.

The immunosuppressive microenvironment of hematological malignancies can attenuate CAR-T anti-tumor activity and persistence through various mechanisms, constituting an important factor in CAR-T resistance or relapse [48–50]. Several studies have reported that high baseline levels of immunosuppressive cells, such as regulatory T cells (Tregs), myeloid-derived suppressor cells, and inhibitory neutrophils, in R/R B-ALL patients are associated with poor outcomes [51–54], suggesting that these inhibitory cells are potential therapeutic targets. Furthermore, checkpoint ligand–receptor interactions result in the delivery of coinhibitory signals, and targeting immune checkpoints may improve CAR-T efficacy [55], although evidence in B-ALL remains limited.

Adverse events

CRS

Tocilizumab and corticosteroids are the mainstays of cytokine release syndrome (CRS) management. Tocilizumab effectively controls CRS without compromising in vivo CAR-T expansion, response rates, or long-term outcomes in B-ALL patients [56]. The early use of tocilizumab upon onset of mild CRS symptoms, or prophylactic use in high-risk populations, may mitigate severe CRS risk [57, 58].

Corticosteroids are used for CRS that does not improve after tocilizumab, although concerns exist regarding the potential impairment of CAR-T efficacy [59–61]. Several studies have shown that corticosteroid use [62–64], even when initiated early [57, 65], does not affect CAR-T expansion or efficacy in B-ALL patients. Considering the additional negative effects of steroids (e.g., increased infection risk), minimizing the dose and duration of corticosteroid therapy to the lowest effective level while ensuring efficacy is recommended [66]. For refractory CRS, options such as the IL-6 inhibitor siltuximab [67], the IL-1 receptor antagonist anakinra [68] and the JAK inhibitor ruxolitinib [69] can be considered, but the evidence is limited.

Research efforts are also directed at mitigating CRS and other toxicities at their source, including optimized CAR-T infusion protocols and the design of less toxic CAR-T constructs. In the FELIX study, investigators adjusted the dosing regimen on the basis of bone marrow disease burden and administered obe-cel in fractions (Days 1 and 10), ultimately reporting only 3 cases (2.4%) of ≥grade 3 CRS and 9 cases (7.1%) of ≥grade 3 immune effector cell-associated neurotoxicity syndrome (ICANS) [3]. In the phase 2 CART19-BE-02 trial, varnimcabtagene autoleucel (var-cel) was administered four escalating fractions (0.1, 0.3, 0.6, and 2 × 10⁶ CAR-T cells per kg). Among 32 adults with R/R B-ALL, the incidence of grade ≥ 3 CRS was only 12%, and that of any-grade ICANS was 3% [70]. These findings highlight the potential of personalized dose adjustment and fractionated administration to increase CAR-T safety.

The design of low-toxicity CAR-T cells involves modifications to the CAR structure and genetic engineering of CAR-T cells. Obe-cel utilizes a low-affinity scFv, contributing to reduced toxicity. As mentioned previously, obe-cel demonstrated favorable safety in the FELIX trial [3]. Strategies such as engineering CAR-T cells to overexpress membrane-bound receptors or to secrete cytokine blockers can help neutralize circulating proinflammatory cytokines [71, 72]. In addition, a phase I trial of ssCART-19 cells, an anti‑CD19 CAR‑T engineered with IL‑6‑targeting short hairpin RNA, reported a grade ≥ 3 CRS in 17.6% of patients and no ICANS, with a 3-month objective response rate (ORR) of 64.7%, suggesting that IL‑6 knockdown may enhance safety without compromising efficacy [73]. Future head‑to‑head trials are needed to validate the superiority of ssCART-19 cells over conventional CAR‑T in terms of safety.

Neurotoxicity (ICANS)

The risk factors for ICANS overlap with those for CRS, including high disease burden, high CAR-T dose, high peak expansion, intensive lymphodepletion, and elevated proinflammatory cytokines and endothelial injury markers. The severity of CRS is also correlated with ICANS [74, 75]. Unique predictors include preexisting neurological comorbidities [74], and biomarkers such as neurofilament light chain (NfL) and glial fibrillary acidic protein, which reflect axonal injury and ICANS severity [76, 77].

The standard treatment for ICANS is corticosteroid therapy. However, there is no standardized management for corticosteroid-refractory ICANS. Given the shared pathophysiology between CRS and ICANS, some strategies used for refractory CRS management can be adapted for use in refractory ICANS. The IL-1 receptor antagonist anakinra is often considered a first-choice treatment for refractory ICANS, as it has been shown in multiple retrospective studies to be effective without compromising CAR-T efficacy [78, 79]. In a prospective study by Park et al., prophylactic anakinra administration also reduced the incidence of ICANS in lymphoma patients [80]. Siltuximab has also demonstrated efficacy in recent retrospective studies [67, 81], but support from prospective data.

Hematotoxicity

Immune effector cell-associated hematotoxicity (ICAHT) refers to cytopenias that occur after CAR-T-cell therapy [82]. ICAHT pathogenesis is multifactorial, stemming from baseline hematopoietic reserve, prior therapies, lymphodepletion, and CAR-T-mediated bone marrow inflammation [83–85].

Multiple risk factors have been associated with ICAHT. These have been detailed in other reviews [82, 85]. Additionally, Nair et al. [86] recently developed the ALL-Hematotox (ALL-HT) scoring system to predict hematologic toxicity post-CAR-T in R/R B-ALL patients. The system incorporates baseline bone marrow disease burden, CRP level, absolute neutrophil count, platelet count, and hemoglobin level. The ALL-HT score effectively predicted the occurrence of severe neutropenia in B-ALL patients [area under the curve (AUC) = 0.84]. [86] Liang et al. [87] also developed two predictive models for early severe ICAHT: eIPM Pre (based on preinfusion factors) and eIPM Post (which replaces preinfusion ferritin with Day 3 post-CAR-T ferritin). Their AUCs in R/R B-ALL patients were 0.87 and 0.88, respectively, indicating good discriminatory ability. [87] Identifying risk factors and applying predictive models can facilitate early recognition of ICAHT patients, enabling subsequent management and reducing nonrelapse mortality.

Treatment for ICAHT remains exploratory. On the basis of existing experience, transfusion therapy and hematopoietic growth factors are potential options [85]. Granulocyte colony-stimulating factor (G-CSF) is typically used to shorten the duration of neutropenia after treatment with CAR-T [82]. Early/prophylactic G-CSF in lymphoma patients has been shown to safely and effectively reduce the incidence of neutropenia or accelerate neutrophil recovery [88, 89], but these benefits were not observed in B-ALL patients [90]. Given that this evidence is based on retrospective studies, prospective clinical trials are needed to investigate the safety and optimal dosing strategy of G-CSF. For refractory ICAHT, low-dose corticosteroids [91, 92] and high-dose intravenous immunoglobulin (IVIG) [93] may be effective, but the evidence is limited. If a donor is available, infusion of donor hematopoietic stem cells to restore hematopoiesis is another option, although the optimal cell dose and timing require further determination [94].

Hypogammaglobulinemia

In B-ALL patients, hypogammaglobulinemia (HG) is a manifestation of the on-target, off-tumor toxicity of CD19 CAR-T therapy [95]. HG signifies humoral immune deficiency, which significantly increases the risk of infection [96]. Current management relies on serum IgG monitoring and immunoglobulin replacement, with more aggressive strategies typically reserved for pediatric patients [97].

IEC-HS

Some patients receiving CAR‑T therapy develop hemophagocytic lymphohistiocytosis (HLH)-like conditions, termed immune effector cell‑associated hemophagocytic lymphohistiocytosis (IEC‑HS), which can cause severe multiorgan dysfunction [98]. Although IEC-HS often occurs during or after CRS resolution and shares overlapping pathogenesis and clinical features with CRS, it is considered a distinct entity requiring different treatment approaches [98]. These findings underscore the need for careful differentiation between severe CRS and IEC-HS, where serum cytokine profiles and ferritin levels may aid in distinction [99, 100].

Treatment for IEC-HS also lacks data from prospective studies. The currently recommended first-line therapy is anakinra±corticosteroids, which are initiated upon the development of grade 2 IEC-HS. If no improvement occurs, anakinra and corticosteroids are combined at increased doses, and second-line treatment with ruxolitinib may be considered [98]. For IEC-HS that is refractory to second-line therapy, emapalumab can be used, and case reports have reported its efficacy in controlling refractory IEC-HS [101, 102].

Accessibility concerns

Despite the approval of multiple CAR‑T products for R/R B‑ALL, the transformative potential of this therapy remains limited by a series of accessibility barriers that prevent many eligible patients from receiving it. These barriers span patient-specific, logistical, and financial domains. Understanding these challenges is critical for developing strategies to broaden the reach of CAR-T therapy.

All currently approved CAR-T products are derived from autologous T cells. However, considering patient-specific factors, autologous CAR-T therapy has several limitations. First, some patients may not successfully undergo apheresis because of severe lymphopenia or clinical instability, all of which can preclude the collection of sufficient autologous T cells. Second, even when apheresis is feasible, the quality of harvested T cells may be compromised by prior treatments and the tumor microenvironment [103]. These limitations restrict the applicability of autologous CAR‑T in certain patients and underscore the urgent need for allogeneic alternatives.

The conventional manufacturing process for autologous CAR-T takes 2–4 weeks from apheresis to product release and does not include additional time for transportation or quality control [104]. For patients with rapidly progressive R/R B-ALL, this waiting period is associated with a substantial risk of disease progression or preinfusion death, which may render them ineligible for infusion [1]. In addition, prolonged vein‑to‑vein time (from apheresis to infusion) has been found to be associated with significantly lower CR rates and reduced OS [105]. These findings underscore the urgent need for faster CAR‑T manufacturing and more efficient logistics, which require innovative manufacturing strategies such as rapid point-of‑care (POC) production or the use of off‑the‑shelf allogeneic products to shorten the vein‑to‑vein time.

Finally, the high cost of CAR‑T therapy represents another significant barrier to accessibility. Owing to the personalized and complex manufacturing process, the cost of approved commercial CAR‑T products exceeds $400,000 per infusion [106], which imposes a heavy economic burden on patients and healthcare systems. This high cost also exacerbates global access disparities, as these products remain largely unavailable in low‑ and middle‑income countries and are often restricted to only a fraction of eligible patients even in high‑income settings [107]. Therefore, reducing the cost of CAR‑T therapy is an urgent need, which may be pursued through optimizing manufacturing processes, such as POC production. Notably, the academically developed var‑cel in Spain has demonstrated that hospital‑based manufacturing can lower costs while maintaining efficacy [70], and emerging in vivo CAR‑T generation strategies hold further promise for simplifying production and reducing expenses [108].

Extramedullary disease

The inherent migratory biology of B-ALL confers the ability to infiltrate extramedullary sites, often involving protected sanctuaries such as the CNS and testes, leading to extramedullary disease (EMD) [109]. Early studies, such as the ELIANA and ZUMA-3 trials, excluded patients with CNS-3 status (defined as ≥ 5 white blood cells/µL in cerebrospinal fluid with identifiable lymphoblasts) [1]. However, as research has advanced, subsequent studies have increasingly demonstrated that CAR-T cells can effectively migrate into the CNS and eliminate leukemic blasts, resulting in a CR in 71%-95% of patients with relapsed/refractory (R/R) B-ALL and CNS involvement, which is comparable to that observed in patients with medullary disease [110–112].

Data on CAR-T therapy for non-CNS EMD are relatively limited. Previous case reports have described the use of CAR-T in the treatment of cutaneous and testicular infiltrates in B-ALL patients [113, 114], indicating that CAR-T cells also possess migratory and antitumor activity against non-CNS EMD. However, the response patterns of non-CNS EMD to CAR-T differ from those of medullary disease. Holland et al. [115] observed disparities in the timing and depth of response to CAR-T between patients with medullary disease and non–CNS EMD: among 18 R/R B-ALL patients with non-CNS EMD, three (16.7%) required 3–6 months to achieve the best response in extramedullary sites, which was significantly later than the best response typically seen in marrow disease by Day 28. Among the 14 patients who achieved CR in the bone marrow, seven did not achieve CR in their non-CNS EMD lesions. Similarly, Epperly et al. [116] also reported delayed responses or a suboptimal depth of remission in non-CNS EMD following CAR-T therapy. These differences may be attributed to variations in CAR-T-cell trafficking to EMD sites and the influence of the EMD microenvironment. These findings suggest inadequate CAR-T infiltration into EMD sites or a more immunosuppressive microenvironment in EMD than in bone marrow. Future studies should focus on elucidating the activity and kinetics of CAR-T in EMD and developing strategies to enhance efficacy against EMD, particularly non-CNS EMD.

Advances in CAR-T therapy for B-ALL

Optimization of CAR-T via CRISPR technology

In the context of CAR-T therapy for R/R B-ALL, the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to knock out specific genes in CAR-T cells can enhance therapeutic outcomes across multiple dimensions, including efficacy, safety, and accessibility (Fig. 2). One of the core strategies to improve efficacy involves the targeted knockout of inhibitory molecules such as immune checkpoints on the surface of CAR-T cells. For instance, Agarwal et al. [117] generated cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)-KO CAR-T cells, which significantly reduced tumor burden and prolonged survival in a B-ALL mouse model. Additionally, compared with immune checkpoint blockade or knockout alone, the CRISPR/Cas9-mediated knockout of key regulators of T-cell exhaustion in CAR-T cells may reshape T-cell fate more effectively, conferring more durable resistance to exhaustion [118, 119]. These modified CAR-T cells maintain an early phenotype and proliferative capacity under persistent antigen exposure, demonstrate prolonged survival and enhanced antitumor efficacy in various tumor mouse models, including B-ALL, and exhibit potential for clinical application.

Fig. 2.

Optimizing the application of CAR-T in B-ALL through CRISPR knockout, knock-in, and knockout screening. B-ALL: Acute B-lymphoblastic leukemia; DSB: Double-strand breaks; HDR: Homology-directed repair; Indels: Insertions or deletions; KI: Knock-in; KO: Knock-out; NHEJ: Nonhomologous end joining; sgRNA: Single-guide RNA. Figure created with Biorender

In terms of safety, the primary concerns are CRS and ICANS. A major strategy involves the use of CRISPR/Cas9 to knock out key cytokines in CAR-T cells involved in CRS/ICANS while avoiding the impairment of CAR-T efficacy [120, 121].

The improvement in CAR-T accessibility via CRISPR is best exemplified by the use of universal CAR-T (UCAR-T) cells derived from healthy donors. CRISPR editing enables these allogeneic T cells to function more safely and persistently in patients. To prevent host immune rejection, UCAR-T cells are typically engineered to disrupt human leukocyte antigen (HLA) class I expression via β2‑microglobulin knockout. Additionally, CD52 knockout allows UCAR-T cells to resist alemtuzumab, which is used in lymphodepletion to clear host lymphocytes while sparing infused UCAR-T cells, thereby increasing their persistence [103].

Beyond gene knockout, CRISPR/Cas9 can also achieve targeted knock‑in (KI) of CAR components via homology‑directed repair [122]. A classic example is the integration of a CD19 CAR into the TRAC locus to generate TRAC-CAR-T cells [123]. This site-specific integration ensures uniform CAR expression, and regulation by the endogenous TRAC promoter maintains optimal CAR levels, reducing exhaustion. Compared with conventional CAR‑T cells with random viral integration, TRAC‑CAR‑T cells achieved more durable disease control in a B‑ALL mouse model [123]. Another strategy is to integrate the CD19 CAR into the PD1 locus (PD1‑CAR‑T). In a small cohort of eight patients with relapsed/refractory lymphoma, seven achieved CR, demonstrating preliminary efficacy [124]. In summary, CRISPR-delivered CAR‑T cells offer advantages such as precise targeting and the ability to simultaneously knock out detrimental genes. However, their long‑term efficacy and safety require further validation in larger patient populations, including those with B‑ALL.

The above gene KO/KI strategies are limited to commonly known targets, whereas CRISPR screening provides a powerful tool for exploring complex regulatory networks within T cells [125]. Many studies have used CRISPR knockout screens to identify critical regulators of T-cell antitumor function [126, 127]. Knockout of these targets enhanced the antitumor function of CAR-T cells in tumor mouse models, including B-ALL, demonstrating clinical potential; however, further validation is needed for clinical translation.

Compared with T-cell/CAR-T screens, CRISPR screens of B-ALL blasts are more disease specific. CRISPR screening of B-ALL blasts can identify factors that mediate sensitivity or resistance to CAR‑T therapy under immune pressure. In B-ALL, the knockout of death receptor pathway genes [128, 129], CD58 (impairing immune synapse formation) [130], or the pro‑apoptotic protein NOXA [131] leads to resistance. Tang et al. [132] identified autophagy as a resistance mechanism. The knockout of autophagy-related genes enhanced CAR‑T killing, and the expression levels of these genes distinguished responders from non‑responders (ROC = 0.776). Key regulators of target antigen expression have also been revealed: SPPL3 knockout causes hyperglycosylation of CD19, hindering CAR recognition [12], whereas NUDT21 knockout increases CD19 expression and CAR‑T efficacy [133]. In the B-ALL microenvironment, knockout of IFNγR/JAK/STAT signaling sensitized B-ALL cells to CAR‑T therapy by blocking HLA‑E upregulation, which otherwise delivers an inhibitory signal via NKG2A/CD94 on CAR‑T cells [134]. These studies using CRISPR screening have revealed various factors influencing the response of B-ALL to CAR-T therapy. Future work should further validate whether these molecules can serve as biomarkers for predicting the efficacy of CAR-T therapy in B-ALL patients, develop drugs targeting these molecules, and investigate their efficacy in combination with CAR-T therapy for resistant populations.

Optimization of cell sources and CAR-T manufacturing

To address the challenges associated with CAR-T accessibility, significant efforts have been made to optimize cell sources and manufacturing processes, aiming to provide infusion opportunities for previously ineligible patients, shorten vein‑to‑vein time, and reduce economic burden. Recent developments include allogeneic CAR‑T products, rapid manufacturing platforms, point-of‑care production, and in vivo CAR‑T generation (Fig. 3).

Fig. 3.

Manufacture of CAR-T Cell Products. A: Allogeneic CAR-T; B: Autologous CAR-T and optimization strategies. cGMP: good manufacturing practice compliant facility; MHC: major histocompatibility complex; POC: point-of-care; TCR: T-cell receptor. Figure created with Biorender

Donor‑derived CAR‑T cells are mainly used for B‑ALL patients who relapse after allogeneic-hematopoietic stem cell transplantation (allo-HSCT). Because these patients have previously reconstituted the donor’s immune system, the risk of rejecting donor-derived CAR-T cells is reduced. In a study by del Bufalo et al., [135] 13 patients received CAR‑T cells from HLA‑matched or haploidentical donors; all achieved MRD‑negative CR, and 8 remained in remission at a median follow‑up of 12 months. Zhang et al. [136] treated 43 post‑transplant patients with donor-derived CD19 CAR‑T: 34 (79%) achieved a complete response (CR), with 1-year event-free survival (EFS) and OS rates of 43%, and only two patients had grade ≤ 2 acute graft-versus-host disease (GvHD). Roddie et al. [137] reported a 93% MRD-negative CR rate (13/14) and further confirmed that lymphodepletion prior to donor CAR‑T infusion improved the 1-year EFS and OS.

Donor-derived CAR-T cells are still patient specific. In contrast, UCAR-T from third-party donors offer off-the-shelf availability. UCAR-T cells use gene editing to disrupt HLA matching requirements and are a major focus in R/R B-ALL research (Table 2). In a study by Benjamin et al. [138]., using TALEN was used to knock out TRAC and CD52, 21 R/R B-ALL patients received UCAR-T. The CR rate was 67% (14/21), with 10 cases of MRD-negative remission. However, UCAR-T persistence was suboptimal: 71% of the patients experienced allo-HSCT, and only 5 patients remained alive at the data cutoff [138]. UCAR-T offers an alternative for patients who are ineligible for autologous CAR-T, with moderate efficacy and safety. However, safety concerns include alemtuzumab-related infection risk, potential GvHD, and off‑target toxicity. Efficacy is limited by poor expansion and durability, possibly because of host immune rejection, and T-cell receptor (TCR) knockout may further compromise persistence [139]. Recently, Wu et al. [140]. identified SPPL3 knockout as a new strategy. SPPL3 KO increases TCR/CD3 glycosylation, preserving TCR expression while reducing the recognition of allogeneic HLA molecules, thus lowering the risk of GvHD without compromising CAR-T effector function. Importantly, the authors confirmed that TCR signaling is crucial for the long-term in vivo persistence and tumor control of CAR-T cells, providing a rationale for their TCR-preserving strategy [140]. In a preliminary study of three patients (including one with B-ALL) receiving SPPL3-KO UCAR-T from HLA-mismatched donors, adverse events were manageable, no typical GvHD occurred, and the B-ALL patient achieved MRD-negative CRi with good CAR-T expansion and persistence [140].

Table 2.

Published clinical trial data on allogeneic CAR-T for R/R B-ALL

Reference	Trial ID	Number of patients	Target	Genetic modification	Dose	LD	CR/CRi	CRS/ sCRS	ICANS/ sICANS	GvHD	Long term outcomes
Donor-derived CAR-T
del Bufalo et al. [135]	NA	13	CD19	-	DL1: 10^6/kg, DL2: 2*10^6/kg, DL3: 3*10^6/kg	Flu + Cy	13 (13 MRD negative)	10/0	1/0	1	With a median follow-up of 12(5–21) months, 8 of 13 patients (62%) maintained CR
Zhang et al. [136]	ChiCTR-OOC-16,008,447, ChiCTR-OIC-17,012,374	43	CD19	-	0.4–12*10^6/kg	Flu + Cy (n = 34), Flu + Cy + Ara-C(n = 5), C(n = 4)	34	38/7	9/0	2	1-year EFS: 43%; 1-year OS: 43%; 1-year CIR: 41%(in patients achieving CR not receiving a second transplant)
Luo et al. [141]	NCT04516551	9	CD19	-	DL1: 6*10^5/kg, DL2: 2*10^6/kg	Flu + Cy	9 (9 MRD negative)	9/1	0	3	1-year PFS: 77.8%; 1-year OS: 85.7%
Roddie et al. [137]	NCT02893189	14	CD19	-	DL1: 10^6/kg, DL2: 3*10^6/kg, DL3: 10^7/kg	Flu + Cy (n = 7), No LD(n = 7)	13 (13 MRD negative)	6/1	0	2	1-year EFS: 21.4%; 1-year OS: 54.6%; 1-year EFS: 28.6%(in patients receiving LD); 1-year OS: 83.3%(in patients receiving LD); 1-year EFS: 14.3%(in patients without receiving LD); 1-year OS: 28.6%(in patients without receiving LD)
Deng et al. [142]	NA	14	CD19(n = 11), CD22(n = 1), CD19 + CD22(n = 2)	-	NA	Flu + Cy	11	14/2	2/2	3	1-year OS: 56%; 2-year OS: 44%
Lussana et al. [143]	NCT03389035, NCT05252403	36	CD19	Sleeping Beauty transposon	> 7.5*10^6/kg	Flu + Cy	30 (24 MRD negative)	15/0	1/0	0	1-year EFS: 32%; 1-year OS: 57%; Median DOR: 8.1 months
Off-the-shelf CAR-T
Benjamin et al. [138]	NCT02808442, NCT02746952	21(14 adults and 7 children)	CD19	TALEN KO TRAC and CD52	Children: 1.1–2.3*10^6/kg; Adults: DL1: 6*10^6, DL2: 6–8*10^7, DL3: 1.8–2.4*10^8	Flu + Cy+ Alemtuzumab (n = 17), Flu + Cy (n = 4)	14 (10 MRD negative)	19/3	8/0	2	6 months RFS: 33%; 6 months OS: 55%; Median DOR: 4.1 months
Ottaviano et al. [144]	NCT04557436	6	CD19	CRISPR/Cas9 KO TRAC and CD52	0.8-2.0*10^6/kg	Flu + Cy+ Alemtuzumab	4 (4 MRD negative)	6/0	2/1	1	Two patients remain in remission over 12 months and 3 months after allo-HSCT, respectively
Hu et al. [145]	NCT04227015	6	CD19 + CD22	CRISPR/Cas9 KO TRAC and CD52	DL1: 10^6/kg, DL2: 3*10^6/kg	Flu + Cy+ Alemtuzumab	5 (5 MRD negative)	6/1	0	0	With a median follow-up of 4.3(2–8) months, 3 of the 5 patients (60%) remained MRD negative
Sun et al. [146]	NCT06307600	4	CD19	TRAC KO, Protein Z overexpression	DL1: 10^5/kg, DL2: 3*10^5/kg, DL3: 5*10^5/kg	Flu + Cy	3 (3 MRD negative)	2/0	0	0	NA
Jain et al. [147]	NCT03666000	15	CD19	Knocking CD19 CAR into TRAC gene locus via ARCUS nuclease	DL1: 3*10^6/kg, DL2: 5*10^8/kg	Flu + Cy	9 (5 MRD negative)	9/0	4/1	0	NA
Jain et al. [148]	NCT04150497	3	CD22	TALEN KO TRAC and CD52	10^6/kg	Flu + Cy / Flu + Cy+ Alemtuzumab	2 (2 MRD negative)	2/0	0	NA	NA
Li et al. [149]	NCT05105867	4	CD19 + CD7	TRAC and CD7 KO	DL1: 10^7/kg, DL2: 1.5*10^7/kg	Flu + Cy	3	4/2	0	0	NA
Hu et al. [150]	NCT05350787	8	CD19	Intracellular retention of TCR/HLA-I	DL1: 3*10^6/kg, DL2: 5*10^6/kg	Flu + Cy +Etoposide	7 (7 MRD negative)	8/2	3/2	NA	With a median follow-up of 146(56–407) days, 4 of 7 patients (57%) remained MRD-negative
Shahid et al. [151]	NCT01430390	6	CD19	EBV specific CAR-T without gene editing	DL1: 10^6/kg, DL2: 3*10^6/kg, DL3: 10^7/kg	Flu/ Flu + Cy /Flu+Clofarabine	3	1/0*	3/0*	3*	36 months EFS: 33%; 36 months OS: 33%

CR: Complete remission; CIR: Cumulative incidence of relapse; CRISPR: Clustered Regularly interspaced short palindromic repeats; CRS: Cytokine release syndrome; Cy: Cyclophosphamide; DL: Dose level; DOR: Duration of remission; EBV: Epstein-Barr virus; EFS: Event free survival; Flu: Fludarabine; HLA: Human leukocyte antigen; ICANS: Immune effector cell associated neurotoxicity syndrome; KO: Knock-out; LD: Lymphodepletion; MRD: Minimal residual disease; NA: Not applicable; PFS: Progression free survival; OS: Overall survival; RFS: Relapse free survival; sCRS: Severe cytokine release syndrome; sICANS: Severe immune effector cell associated neurotoxicity syndrome; TALEN: Transcription activator-like effector nuclease; TCR: T cell receptor

* Including B cell lymphoma patients

In addition to gene-editing strategies for UCAR-T production, non-gene-editing strategies also exist. For example, engineering CAR-T cells to express an anti-CD3-scFv with a KDEL tag can prevent TCR/CD3 secretion from the endoplasmic reticulum, thereby reducing surface TCR expression. This non-gene-edited UCAR-T therapy was preliminarily tested in 8 patients with R/R B-ALL, 7 of whom achieved MRD-negative CR/CRi [150]. Another non-gene-editing strategy for manufacturing UCAR-T involves the use of virus-specific T cells to construct CAR-T cells. Unlike conventional CD3-activated T cells, virus-specific T cells primarily recognize viral antigen epitope-HLA complexes through their TCR, thereby reducing their alloreactivity and risk of GvHD [151, 152]. Recent results from a phase 1 study of off-the-shelf Epstein–Barr virus-specific CAR-T cells were reported. Among 6 patients with R/R B-ALL, 3 achieved or maintained CR. However, the persistence of CAR-T cells in this study was poor, potentially related to the enrichment of terminally differentiated T cells in the manufactured product [151].

In addition to commonly used αβ T cells, owing to their inherent antitumor activity and low risk of GvHD, certain specialized subsets of T cells are candidate sources for optimizing UCAR-T therapy. One such unique T-cell subset is γδ T cells, which recognize antigens in a major histocompatibility complex (MHC)-unrestricted manner. This characteristic gives them natural advantages in terms of tumor recognition/clearance and a reduced risk of GvHD [153]. Similarly, invariant natural killer T (iNKT) cells are another rare subtype of T cells characterized by the expression of an invariant TCR [154]. A study using allogeneic CAR-iNKT cells treated 9 patients with B-cell malignancies, among whom 3 achieved CR/CRi [155]. The low risk of GvHD associated with γδ T and iNKT cells as off-the-shelf CAR-T products could reduce the reliance on gene editing. However, the scarcity of these T-cell subsets poses a challenge in the collection of sufficient numbers of cells.

Compared with limited special T-cell subsets, umbilical cord blood (UCB) can provide a more abundant source for UCAR-T manufacturing. Using UCB as a T-cell source offers several advantages: less stringent HLA matching requirements, resulting in increased universality; low immunogenicity and a reduced risk of GvHD; and enrichment of early differentiated UCB T cell phenotypes, conferring stronger proliferative capacity and delayed exhaustion characteristics [156]. Numerous preclinical studies have utilized donor UCB to prepare CAR-T cells, which have demonstrated antitumor activity in B-ALL models [157, 158]. Additionally, induced pluripotent stem cells (iPSCs) can be important sources of raw CAR-T-cell materials. iPSCs possess self-renewal capacity, ensuring cell supply. Genetic manipulations performed in iPSCs (e.g., CAR gene insertion and TRAC knockout) can be passed on to progeny cells during iPSC proliferation, avoiding repeated editing operations and enhancing cell homogeneity [159]. This approach shows promise for clinical application.

To accelerate production, Gracell Therapeutics developed fast CAR-T (F-CAR-T), which completes T-cell isolation, activation, and transduction within one day, omitting the expansion step [160]. In a study of 25 R/R B-ALL patients, the median time from apheresis to infusion was 14 days. All 25 achieved CR/CRi, and among the 20 patients who received consolidative allo-HSCT, 15 (75%) achieved MRD-negative CR at a median follow-up of 693 days [160]. A recent retrospective study revealed that compared with conventional CAR-T, F-CAR-T led to significantly higher CR and MRD negativity rates but no difference in 1- or 2-year leukemia-free survival (LFS)/OS. However, F‑CAR‑T was associated with higher incidences of CRS (91.3% vs. 66.7%) and ICANS (30.4% vs. 9.5%) [161]. These findings suggest that skipping ex vivo expansion reduces cell yield but enables the rapid production of less differentiated CAR‑T cells that expand well in vivo and achieve high response rates at low doses (10⁴–10⁵ cells/kg). However, cryopreservation after activation may render these cells hyperactive [104], explaining the increased toxicity. Thus, rapidly manufactured CAR‑T-cell therapies warrant closer monitoring for adverse events, and their long‑term efficacy requires further evaluation.

POC CAR-T cells constitute another strategy for shortening the manufacturing time and reducing cost. Unlike traditional manufacturing, which requires long-distance cell transport and cryopreservation, POC CAR-T uses automated systems at the treatment site to produce fresh, non-cryopreserved products, reducing both waiting time and cost [104, 162]. In a study by Maschan et al. [163]., CD19 CAR-T cells were manufactured on the CliniMACS Prodigy platform at local sites for 54 patients with B‑cell malignancies. The median time from apheresis to infusion was 13 days. Among 27 evaluable R/R B-ALL patients, 24 (89%) achieved MRD‑negative CR. For those who responded, the 1-year progression-free survival (PFS) and OS were 37.5% and 79.2%, respectively, with a median duration of response (DOR) of 310 days [163]. The VELCART trial in India [164] used the same platform in 10 patients (6 with R/R B-ALL). The vein‑to‑vein time was 9 days; all 6 B-ALL patients achieved CR, and 4 remained alive at the data cutoff [164]. Thus, POC CAR-T cells offer a time- and cost-saving platform for B-ALL. However, widespread adoption faces challenges, including uniform quality control across sites, harmonized regulatory frameworks, and significant investment for scaling [104, 165].

In vivo CAR-T constitutes an emerging strategy that systemically delivers CAR-encoding vectors (e.g., viral vectors or nanoparticles) targeted to T cells, enabling de novo CAR expression directly in the patient. This approach bypasses ex vivo activation and expansion, significantly reducing the manufacturing time. Vectors can be preproduced and stored, allowing immediate administration. In vivo CAR-T cells may also undergo more physiological activation, potentially reducing exhaustion [108]. ESO‑T01, an in vivo lentiviral vector encoding the B-cell maturation antigen CAR and targeted to T cells via a TCR‑specific nanobody, was evaluated in two independent studies. In a case series of 4 R/R multiple myeloma (MM) patients, the ORR was 100% [2 CR, 2 partial remission (PR)], with grade 3 CRS in 3 patients [166]. In a phase I trial of 5 R/R MM patients, the ORR was 80% (3CR, 1 PR) [167]. Adverse events were manageable in both cohorts, and CAR‑T expansion kinetics were comparable to those of commercial products. These preliminary MM results validate the feasibility of the in vivo CAR-T approach and highlight its potential for application in other diseases, such as B-ALL. Future studies with larger cohorts and longer follow-up periods are needed to explore the efficacy and safety of in vivo CAR-T therapy in B-ALL and other indications.

Combination strategies to enhance CAR-T efficacy in B-ALL

Despite the impressive initial efficacy of CAR‑T therapy in R/R B‑ALL, relapse remains a major challenge because of antigen escape, limited CAR-T persistence, and an immunosuppressive microenvironment. To address these challenges, combination strategies have been developed to enhance CAR‑T function, broaden antigen recognition, or remodel the tumor microenvironment. In this section, we review recent advances in dual‑targeting CAR‑T, immune checkpoint inhibitors, small molecule inhibitors, and cytokine armoring strategies (Fig. 4).

Fig. 4.

Combination strategies for improving CAR-T efficacy in B-ALL. A: Overcoming antigen escape; B: Reshaping the immunosuppressive microenvironment; C: Enhancing CAR-T-cell function and persistence; D: Sensitizing B-ALL to CAR-T therapy and synergistic killing. AKTi: Protein kinase B inhibitor; B-ALL: Acute B-cell lymphoblastic leukemia; BT: Bridging therapy; BTKi: Bruton’s tyrosine kinase inhibitor; DC: Dendritic cell; DNMTi: DNA methyltransferase inhibitor; HDACi: Histone deacetylase inhibitor; ICI: immune checkpoint inhibitor; KO: Knock-out; LD: Lymphodepletion; SKI: Phosphatidylinositide 3-kinase inhibitor; sh-RNA: Short-hairpin RNA; TKI: Tyrosine kinase inhibitor; TRUCK: T cells redirected for universal cytokine-mediated killing; VEGF: Vascular endothelial growth factor. Figure created with Biorender

Dual-targeting CAR-T

Dual‑targeting CAR‑T cells recognize two antigens to prevent relapse from single‑antigen loss. In R/R B‑ALL, CD19‑CD22 is the most common approach, with strategies including the sequential administration or co‑administration of two CAR‑T products [168–176] or engineered dual‑targeting CAR‑T cells (cotransduction, bicistronic, or bivalent vectors) (Table 3) [145, 177–188]. Currently, there is no consensus on the optimal strategy. A retrospective study revealed no significant differences in response or 2-year OS between tandem therapy and sequential therapy, although 1-year LFS was more favorable for tandem therapy (71.1% vs. 52.2%, P = 0.154) [184]. However, tandem CD19/CD22 CAR-T cells demonstrate relatively poor recognition efficiency for the CD22 antigen [179, 181], potentially because of steric hindrance between the tandem scFvs [189]. The use of cotransduced CAR-T or bicistronic CAR-T can increase CD22 recognition and prevent escape mediated by CD22 downregulation [181, 185], but these strategies increase the number of CAR molecules expressed per CAR-T cell, potentially leading to activation-induced cell death or exhaustion [181, 185]. The choice of costimulatory domains also matters: a preclinical study showed that the CD19.BBζ‑CD22.28ζ pairing yielded the most durable efficacy, as CD28 enhances unstable CD22 recognition, while 4‑1BB promotes the persistence of the stable CD19 CAR [190]. Overall, despite targeting an additional antigen, dual-targeting therapies do not appear to significantly increase treatment-related adverse events [191–193]. Treatment failures are primarily due to CD19 + CD22+ relapse (insufficient persistence) and, secondarily, CD19-CD22 + relapse (inadequate CD22 targeting). Future efforts should focus on improving persistence and reducing antigen‑negative relapse. In addition to CD22, numerous alternative targets are available, including natural killer Group 2 member D ligand [194], B-cell activating factor receptor [195], and FMS-like tyrosine kinase 3‌.[196] These CD19-X dual-targeting CAR-T constructs have demonstrated potent anti-B-ALL activity in preclinical studies, but their efficacy needs further validation in clinical trials.

Table 3.

Clinical trial data on CD19/CD22 CAR-T for R/R B-ALL

Reference	Trial ID	Number of B-ALL patients infused with CAR-T	CR/CRi rate	CRS/sCRS	ICANS/sICANS (neurotoxicity)	Relapse number and phenotypes* (double positive/CD19neg/CD22neg/double negative/unknown)	Long term outcomes	Dual-target design
Wang et al. [168]	ChiCTR2000032211	225 (including 31 pts with isolated EMD)	99% (192/194, 192 MRD negative); 100% (31/31) for patients with isolated EMD	198/64	47/9	43 (24/16/0/1/2)	12-month EFS: 73.5%; 12-month OS: 87.7%	1:1 co-administration CD19 CAR-T and CD22 CAR-T
Frey et al. [176]	NA	13	100% (11/11, 11 MRD negative)	11/0	2/1	1 (0/0/0/0/1)	With a median follow up of 6.2 mo, 10 patients remained in MRD negative remission	Co-administration humanized CD19 CAR-T and humanized CD22 CAR-T
Wang et al. [169]	ChiCTR-OPN-16,008,526	51	96% (48/50, 48 MRD negative)	85/19**	12/1**	24 (23/0/0/1/0)	Median PFS: 13.6 mo; Median OS: 31.0 mo; 12-month PFS: 52.9%; 12-month OS: 62.8%	Sequential infusion of CD19 CAR-T and CD22 CAR-T
Pan et al. [171]	NCT04340154	81 (including 2 pts only receiving CD19 CAR-T)	98% (79/81, 79 MRD negative)	65/15	26/4	15 (11/2/0/1/1)	18-month EFS: 79%; 18-month DFS: 80%; 18-month OS: 96%; 18-month DOR: 80%	Sequential infusion of CD19 CAR-T and CD22 CAR-T, with a median interval of 39 days between the two infusions
Zhang et al. [172]	ChiCTR1900025419	4	100% (4/4, 4 MRD negative)	3/1	1/0	3 (1/0/0/2/0)	12-month RFS: 75%; 12-month OS: 75%	Sequential infusion of CD22 CAR-T and CD19 CAR-T on day 1 and 2, respectively
Liu et al. [173]	ChiCTR-ONC-17,013,648	27 (including 6 pts only receiving CD19 CAR-T)	85% (23/27) after 1st infusion; 95% (20/21) after 2nd infusion	23/7 after 1st infusion; 11/0 after 2nd infusion	3/1 after 1st infusion; 0 after 2nd infusion	7 (4/2/0/0/1)	12-month EFS: 67.5%; 12-month OS: 88.5% 18-month EFS: 67.5%; 18-month OS: 88.5%	Sequential infusion of CD19 CAR-T and humanized CD22 CAR-T, with a median interval of 2.7 months between the two infusions
Yang et al. [174]	ChiCTR2100053871	29 (including 6 pts only receiving CD19 CAR-T)	100% (23/23, 23 MRD negative) after 2nd infusion	18/2 after 1st infusion; 9/0 after 2nd infusion	2/0 after 1st infusion; 0 after 2nd infusion	8 (6/1/0/1/0)	1-year LFS: 67.1%; 1-year OS: 91.3% 2-year LFS: 47.0%; 2-year OS: 58.6%	Sequential infusion of CD19 CAR-T and CD22 CAR-T, with a median interval of 3.8 months between the two infusions
Annesley et al. [180]	NCT03330691	12	91% (11/12, 11 MRD negative)	5/0	5/1	NA	NA	Co-transduction of CD19 and CD22 CAR vector
Ghorashian et al. [185]	NCT02443831	12	83% (10/12, 10 MRD negative)	11/0	6/1	5 (5/0/0/0/0)	6-month EFS: 75%; 6-month OS: 75% 12-month EFS: 60%; 12-month OS: 75%	Co-transduction of CD19 and CD22 CAR vector
Dai et al. [177]	NCT03185494	6	100% (6/6, 6 MRD negative)	6/0	0	3 (2/0/0/1/0)	Three patients remained in remission for over 8 mo	Bivalent CD19/CD22 CAR-T
Spiegel et al. [178]	NCT03233854	17	88% (15/17, 15 MRD negative)	29/2**	14/4**	10 (4/4/0/0/2)	Median PFS: 5.8 mo; Median OS: 11.8 mo	Bivalent CD19/CD22 CAR-T
Niu et al. [183]	NCT03919526	15 (including 11 pts using as first line consolidation)	100% (15/15, 15 MRD negative)	4/0	1/0	5 (4/1/0/0/0)	12-month RFS: 77%; 12-month OS: 86%	Bivalent CD19/CD22 CAR-T
Shalabi et al. [181]	NCT03448393	20	60% (12/20, 12 MRD negative)	10/3	1/1	4 (3/1/0/0/0, CD22 expression not reported)	6-month RFS: 80.8%; 12-month RFS: 57.7%; Median OS: 23.7 mo	Bivalent CD19/CD22 CAR-T
Cui et al. [182]	NCT03614858	47	100% (47/47, 40 MRD negative)	41/8	1/0	12 (10/2/0/0/0)	2-year LFS: 69.2%; 2-year OS: 73.8%	Bivalent CD19/CD22 CAR-T
Ma et al. [187]	NCT04303520	35	83% (29/35)	13/1	0	16 (0/0/0/0/16)	Median PFS: 4 mo; 1-year PFS: 37%; 1-year OS: 62%	Bivalent CD19/CD22 CAR-T
González-Martínez et al. [188]	NCT06709469	10	80% (8/10, 7 MRD negative)	8/3	3/3	4 (0/1/0/2/1)	18-month EFS: 32%; 18-month OS: 70%	Bivalent CD19/CD22 CAR-T
Hu et al. [145]	NCT04227015	6	83% (5/6, 5 MRD negative)	6/1	0	1 (0/0/1/0/0)	With a median follow-up of 4.3(2–8) mo, 3 of the 5 patients (60%) remained MRD negative	Bivalent CD19/CD22 universal CAR-T, CRISPR KO TRAC and CD52
Srinagesh et al. [186]	NCT03233854	9	89% (8/9, 8 MRD negative)	4/0	0	3 (2/1/0/0/0)	12-month PFS: 67%	Bivalent CD19/CD22 CAR-T, with NKTR-255 supplement on day 14
Cordoba et al. [179]	NCT03289455	15	86% (13/15, 13 MRD negative)	12/0	4/0	9 (6/2/0/1/0)	6-month EFS: 48%; 6-month OS: 80% 12-month EFS: 32%; 12-month OS: 60%	Bicistronic humanized CD19/CD22 CAR-T

CR: Complete remission; CRISPR: Clustered Regularly interspaced short palindromic repeats; CRS: Cytokine release syndrome; Cy: Cyclophosphamide; DL: Dose level; DOR: Duration of remission; EBV: Epstein-Barr virus; EFS: Event free survival; EMD: Extramedullary disease; ICANS: Immune effector cell associated neurotoxicity syndrome; KO: Knock-out; MRD: Minimal residual disease; NA: Not applicable; PFS: Progression free survival; OS: Overall survival; RFS: Relapse free survival; sCRS: Severe cytokine release syndrome; sICANS: Severe immune effector cell associated neurotoxicity syndrome; TALEN: Transcription activator-like effector nuclease

* Relapse number (CD19 + CD22+/CD19-CD22+/CD19 + CD22-/CD19-CD22-/unknown phenotype, low level expression is classified as negative)

** Including B cell lymphoma patients

Immune checkpoint inhibitors

Clinical data on the combined use of CAR-T-cell therapy and PD-1/PD-L1 inhibitors for R/R B-ALL remain limited. In a study of four children with poor CAR‑T response or expansion, pembrolizumab prolonged CAR‑T persistence overall and induced objective responses in two children [197]. In another study of 13 children with suboptimal CAR‑T responses, PD‑1 inhibitor infusion restored B‑cell aplasia in three of six children with early B‑cell recovery. Among four patients with extramedullary disease, two achieved CR, and two achieved PR [198]. In addition to PD‑1, CTLA-4 knockout enhances CAR‑T anti‑B‑ALL activity [117], and the inhibition of V-domain immunoglobulin suppressor of T-cell activation synergizes with CAR‑T activity in murine models [199]. Furthermore, a recent study engineered CAR‑T cells to secrete a T-cell immunoglobulin and mucin domain-containing protein 3 (TIM‑3)‑Fc decoy receptor, which protected against TIM‑3/Galectin‑9‑mediated exhaustion and improved antitumor activity and persistence in B‑ALL mice [200].

Small-molecule inhibitors

Dasatinib reversibly blocks CAR/TCR signaling, preventing exhaustion and restoring the function of exhausted CAR-T cells via epigenetic reprogramming. Intermittent dasatinib enhanced the antitumor activity of CAR-T therapy and prolonged survival in B-ALL murine models [201]. Clinically, dasatinib can serve as bridging or consolidation therapy for Ph + B-ALL [202]. In a recent study of newly diagnosed Ph + B-ALL patients, dasatinib-based induction followed by sequential CD19/CD22 CAR-T-cell therapy resulted in a molecular CR of 85% (23/27) and a 2-year LFS/OS of 92% [203]. Additionally, the PI3K/AKT/mTOR pathway regulates T-cell proliferation and differentiation. Pretreating CAR-T cells during manufacture with PI3K inhibitors [204] and AKT inhibitors [205] has been shown in preclinical studies to reduce CAR-T exhaustion, increase the number of less differentiated phenotypes, and thereby enhance in vivo expansion and antitumor activity.

Ibrutinib, a Bruton’s tyrosine kinase inhibitor (BTKi), also inhibits interleukin‑2‑inducible T-cell kinase, which is involved in T-cell differentiation. The addition of ibrutinib during CAR‑T manufacturing increases the number of cells with less differentiated phenotypes and reduces the expression of exhaustion markers [206, 207]. In B-ALL murine models, compared with CAR-T therapy alone, ibrutinib combined with CAR‑T therapy reduced tumor burden and prolonged survival [208, 209]. Furthermore, ibrutinib inhibits transcriptional reprogramming such that low CD19 expression is maintained in B‑ALL blasts, thereby increasing CAR-T-cell cytotoxicity [24]. Additionally, CD19‑negative B‑ALL cells become dependent on the BCR‑BTK‑MEK pathway and are sensitive to BTKis [210], suggesting that combining ibrutinib with CAR‑T may help overcome CD19 antigen escape.

Decitabine can alter exhaustion-related DNA methylation in CAR-T cells. Adding low-dose decitabine (10 nM) during ex vivo CAR‑T-cell culture increases the expression of proliferation‑ and memory‑associated genes, decreases the expression of exhaustion‑related genes, and reduces terminal differentiation. In B-ALL and lymphoma murine models, decitabine-pretreated CAR-T cells exhibited enhanced antitumor activity, persistence, and memory phenotype maintenance [211]. Consistent with preclinical findings, a study in which decitabine was added to the cyclophosphamide (Cy) and fludarabine (Flu) (Cy/Flu) lymphodepletion regimens for R/R B‑ALL patients demonstrated improved long‑term survival in the decitabine + Cy/Flu group compared with that in the conventional lymphodepletion group [212].

Histone deacetylase inhibitors (HDACis) can directly improve CAR‑T function. The class I HDACi chidamide remodels CAR‑T epigenetics and activates the Wnt/β‑catenin pathway, maintaining a memory phenotype and alleviating exhaustion. In B‑ALL murine models, the pretreatment of CAR‑T with chidamide or oral gavage of HDACis after CAR‑T injection enhanced antitumor function, preserved the memory phenotype, and prolonged survival [213]. Additionally, chidamide sensitizes B‑ALL cells to CAR‑T by upregulating CD22 expression [214] and upregulating the expression of the proapoptotic protein NOXA [131], thereby increasing CAR‑T efficacy. Clinically, the use of chidamide as maintenance therapy in 19 R/R B‑ALL patients who achieved remission after CAR‑T therapy yielded a 1-year disease-free survival of 62.9% and a 1-year OS of 83.3% among the 13 patients who did not receive allo‑HSCT [215].

Pretreatment with BH3 mimetics (e.g., venetoclax) upregulates the expression of CD19 and the pro‑apoptotic protein BAK on B‑cell tumors, increasing CAR-T cytotoxicity. However, concurrent or post‑CAR‑T-cell therapy promotes CAR‑T apoptosis and exhaustion, limiting efficacy [216]. To address this, CAR‑T cells engineered to overexpress the antiapoptotic protein Bcl‑xL were shown to resist BH3 mimetic‑induced toxicity, and combining venetoclax with Bcl‑xL‑overexpressing CAR‑T prolonged survival in B‑ALL murine models [217]. In a recent clinical study of newly diagnosed high‑risk Ph‑negative B‑ALL patients, venetoclax plus azacitidine induction followed by CD19/CD22 tandem CAR‑T therapy resulted in a CR rate of 91.7% (11/12), with a 10% MRD-negative outcome. The 1-year cumulative incidence of relapse (CIR), LFS, and OS was 18.2%, 81.8%, and 81.8%, respectively [218].

Cytokines

Interleukin (IL)-2 is typically added during CAR-T manufacturing to increase ex vivo expansion, but it can also promote CAR-T terminal differentiation and Treg expansion [219]. Compared with the use of IL-2, the use of cytokines such as IL-7, IL-15, or IL-21 during manufacturing also promotes CAR-T expansion and leads to the development of fewer differentiated phenotypes [219]. Additionally, the addition of IL-4 during CAR-T generation enhances type 2 immune function. CAR-T cells with high levels of type 2 function showed significantly greater expansion, lower exhaustion, and longer survival in B-ALL murine models than those with low levels of type 2 function did [220]. Systemic cytokine supplementation post‑infusion is being explored. In a phase I study of CD19/CD22 CAR‑T plus the IL‑15 receptor agonist NKTR‑255 in 9 R/R B‑ALL patients, 89% achieved MRD‑negative CR/CRi, and the 1‑year PFS was 67% (vs. 38% in the control group), with only grade 1 CRS occurring in four patients [186]. Beyond systemic administration, fourth-generation CAR-T therapy, also called TRUCK (T cells redirected for universal cytokine-mediated killing), involves genetically modifying CAR-T to enable the local release of proinflammatory cytokines, which act in an autocrine or paracrine manner to improve the tumor microenvironment [221]. CD19 CAR‑T-mediated secretion of IL‑18 in lymphoma patients for whom prior CD19 CAR‑T therapy achieved an ORR of only 81% (17/21) and a CR rate of 52% (11/21), with a safety profile similar to that of conventional CAR‑T therapy [222]. Another strategy involves engineering CAR-T cells to coexpress cytokines; a recent study reported that CD19 CAR‑T cells expressing IL‑10 in 12 R/R B‑ALL patients resulted in 1-month overall remission rates of 100% and 6-month RFS and OS rates of 91% and 100%, respectively [223]. These studies provide clinical proof-of-concept for the feasibility of cytokine-armored CAR-T in cancer therapy, suggesting novel directions for next-generation CAR-T development. Trials involving advanced CAR-T are currently underway in B-ALL and other hematological and solid tumors [221], awaiting further confirmation of their efficacy and safety.

Advances in peri‑CAR‑T management for B-ALL

The successful implementation of CAR‑T therapy also relies on a series of peri‑infusion management steps that critically influence treatment outcomes. As illustrated in Fig. 5, the clinical workflow for R/R B‑ALL patients receiving CD19-directed CAR-T therapy encompasses bridging therapy, lymphodepleting preconditioning, postinfusion monitoring, consolidation, and salvage strategies. In this section, we discuss recent advances and key considerations in each of these peri‑CAR‑T management domains.

Fig. 5.

Management workflow for R/R B-ALL patients receiving CD19-directed CAR-T therapy. AEs: Adverse events; Allo-HSCT: Allogeneic-hematopoietic stem cell transplantation; B-ALL: Acute B-lymphoblastic leukemia; BCA: B-cell aplasia; CR: Complete remission; Cy: Cyclophosphamide; FC: Flow cytometry; Flu: Fludarabine; G-CSF: Granulocyte colony-stimulating factor; GO: Gemtuzumab ozogamicin; HG: hypogammaglobulinemia; ICAHT: Immune cell-associated hematotoxicity; ICANS: Immune effector cell-associated neurotoxicity syndrome; IEC-HS: Immune effector cell-associated hemophagocytic lymphohistiocytosis; Ino: Inotuzumab ozogamicin; IVIG: Intravenous immunoglobulin; MRD: Minimal residual disease; NGS: Next-generation sequencing; NR: No response; PCR: Polymerase chain reaction; TKI: Tyrosine kinase inhibitor; TPO-RA: Thrombopoietin receptor agonist. Figure created with Biorender

Bridging therapy and lymphodepletion

Bridging therapy is used to control disease burden during the CAR-T manufacturing window [224, 225]. In addition to conventional chemotherapy, targeted therapy and immunotherapies can also serve as bridging options. TKIs can be used as bridging regimens for patients with Philadelphia chromosome-positive ALL (Ph + ALL) [202]. Although TKIs have been shown to negatively regulate CAR-T proliferation and activity [226], successful combinations of TKIs and CAR-T have emerged in B-ALL patients [203].

Inotuzumab ozogamicin (InO) is a potential option for bridging therapy in B-ALL patients, resulting in superior tumor burden reduction compared with chemotherapy [227], but it also depletes normal CD19 + B cells, potentially impairing subsequent CD19 CAR-T expansion [19]. Retrospective data have revealed that InO exposure (as bridging or prior therapy) is associated with shorter event‑free survival and overall survival after CAR-T infusion [19, 42, 228, 229]. However, a multivariate analysis suggested that InO exposure was no longer an independent predictor of progression-free survival, indicating that the observed poor outcomes may reflect the selection of a more aggressive, resistant patient population rather than a direct negative effect of InO on CAR-T- function [229]. Given the small sample size of bridging‑specific cases, prospective controlled trials are needed to clarify the impact of InO bridging on CAR-T outcomes.

Blinatumomab shares the same target with CD19 CAR‑T therapy. Prior blinatumomab exposure has been associated with lower response rates and poorer long‑term survival after subsequent CAR‑T therapy in some studies, potentially because of CD19 downregulation or selection of CD19‑negative subclones [21, 230, 231]. However, a large-scale study revealed that only patients who failed to respond to prior blinatumomab had inferior CAR‑T outcomes, whereas blinatumomab responders achieved results similar to those of blinatumomab-naive patients, suggesting that T-cell dysfunction or other resistance mechanisms also contribute [20]. A recent real‑world study in 278 R/R B‑ALL patients also demonstrated that prior blinatumomab nonresponse is correlated with significantly inferior outcomes after brexu-cel therapy [232]. Therefore, the use of blinatumomab as a bridge to CD19 CAR‑T therapy requires careful evaluation.

Lymphodepletion (LD) with Cy/Flu within the week before CAR-T infusion enhances CAR-T expansion and persistence by clearing endogenous lymphocytes, reducing tumor burden, promoting homeostatic cytokines, and mitigating anti-CAR immunity [233]. However, the optimal dosing of LD is not fixed; doses commonly used in clinical trials are a total Flu concentration of 75–120 mg/m² and a total Cy concentration of 750–1500 mg/m², typically given over 1–5 days [233]. Notably, higher Flu exposure (AUC > 13.8–14 mg·h/L) is correlated with better outcomes and lower CD19-positive relapse, whereas lower exposure predicts shorter survival [234, 235]. Given interindividual variability in Flu pharmacokinetics, fixed dosing is suboptimal. A recent study proposed age‑adjusted or pharmacokinetic‑monitored dosing (guided by first‑day Flu levels), which resulted in higher target exposure, improved overall survival, and reduced relapse rates [236]. In summary, existing evidence indicates that the choice of LD regimen and dose, as well as pharmacokinetic monitoring, significantly affects the efficacy of CAR-T therapy for R/R B-ALL. However, while the LD regimen is optimized to achieve the best efficacy, the potential risks of adverse effects such as myelosuppression should also be weighed.

Alternative LD regimens have also been explored. Adding decitabine to Cy/Flu before CD19/CD22 CAR‑T did not improve early MRD negativity but significantly improved 3‑year LFS (92.9% vs. 27.3%) and OS (92.3% vs. 41.7%) [212]. A case report of cladribine plus Cy in a pediatric patient revealed no impairment of subsequent CAR‑T efficacy [237]. With respect to allogeneic CD52-knockout CAR‑T, the addition of alemtuzumab to Cy/Flu enhances CAR‑T expansion and response rates but increases myelosuppression and infection risk [138, 238]. Although lymphodepletion is a standard step in CAR-T therapy, its specific implementation (including drug combination, dosage, and timing) requires further in-depth study to establish individualized LD strategies based on the heterogeneity of B-ALL patients. Finally, emerging in vivo CAR‑T manufacturing may reduce the dependence on LDs, potentially redefining its future role [166].

Monitoring and consolidation therapy

Post-infusion dynamic monitoring is crucial for early relapse detection. Monitoring CAR copy number post infusion is possible. Low peak CAR expansion or a low AUC from Days 0 to 28 is associated with antigen-positive relapse [239, 240], but this method does not reflect CAR-T functional status. More commonly, peripheral blood B-cell levels are monitored. CD19 CAR-T-mediated B-cell aplasia (BCA) reflects the functional persistence of CAR-T cells in vivo, whereas B-cell recovery (BCR) suggests loss of persistence. An international consensus definition of BCA was established to standardize postinfusion monitoring. According to this consensus, a BCA is defined as < 0.01% CD19 + B cells in the bone marrow (out of total white blood cells or mononuclear cells) and 0 CD19 + B cells/µL in the peripheral blood [241]. However, it is important to note that some patients may still have persistent B-cell aplasia at the time of relapse [22, 240], indicating the limitation of monitoring BCA. MRD is another important biomarker for predicting B-ALL relapse and is detectable via flow cytometry, quantitative polymerase chain reaction, or next-generation sequencing (NGS). NGS offers extremely high sensitivity (10^−6), enabling MRD detection early before morphological relapse, thereby providing more time for subsequent interventions. One study revealed that the detection of NGS-MRD positivity in bone marrow samples at 3 months was strongly associated with B-ALL relapse [240]. Therefore, combining peripheral blood B-cell monitoring with MRD assessment is recommended for a more accurate identification of disease relapse [240, 242].

Allo‑HSCT is a key consolidation strategy after CAR‑T-induced remission. However, conflicting reports exist regarding whether bridging to allo-HSCT after CAR-T-cell-induced remission prolongs survival in R/R B-ALL patients (Table 4). Therefore, identifying which types of R/R B-ALL patients are more suitable for and can benefit from bridging allo-HSCT is crucial, whereas the remaining patients may achieve long-term survival after CAR‑T therapy through close monitoring or other less toxic consolidation strategies. When transplantation is feasible, patients with high-risk features for relapse should be identified first. MRD status is among the most important predictors of relapse, and consolidative allo-HSCT should be considered for patients who achieve MRD-positive remission [240, 243]. In the study by Gu et al., [244] consolidative allo-HSCT was associated with improved LFS and OS in patients with MRD-positive remission. Early BCR is another risk factor: Summers et al. [245] reported that for patients with a BCR within 63 days, allo‑HSCT improved LFS. High‑risk genetics also guide decisions: Zhang et al. [246] showed that among TP53 + patients, allo‑HSCT improved 1-year LFS and 1-year OS. Similarly, Jiang et al. [247] reported that patients with low preinfusion MRD (< 5%) but unfavorable cytogenetics or those with high tumor burden (≥ 5%) benefited from allo‑HSCT in terms of both EFS and RFS.

Table 4.

Published studies with outcomes in R/R B-ALL patients receiving post-CAR-T consolidative HSCT or CAR-T alone

Reference	Study design	Median age	CAR-T design	Number of complete responders bridging to HSCT (% of responders)	Disease status prior to HSCT	Previous HSCT (%)	TRM/NRM in HSCT cohort (%)	Relapse in HSCT cohort (%)	Long term outcome in HSCT cohort	Survival/relapse data (HSCT vs. non-HSCT)	Survival data (subgroup comparison)
Park et al. [224]	Prospective	44 (Range: 23–74)	CD19-28z	17 (39%, 17/44)	MRD-CR	19 (36%, all cohort)	6 (35%)	6 (35%)	NA	No significant difference in EFS (P = 0.64) and OS (P = 0.89) among MRD-CR pts; Relapse: 6 (35%) vs. 17 (65%, including relapse and death cases)	NA
Shah et al. [231]	Prospective (ZUMA-3 update)	42.5 (Range: 18–84)	CD19-CD28z (brexu-cel)	14 (24.1%, 14/58)	CR/CRi (n = 14); PR (n = 1)	29 (37%, all cohort)	6 (42.9%)	2 (14%)	Median OS: 36.3 mo (in 14 CR/CRi pts)	Median OS: 36.3 mo vs. 60.4 mo;	NA
Laetsch et al. [248]	Prospective (ELIANA update)	11 (Range: 3–24)	CD19-4-1BBz (tisa-cel)	11 (16.7%, 11/66)	MRD-CR (n = 5); MRD + CR (n = 4); MRD unknown CR/CRi (n = 2); Relapsed disease (n = 6)	6 (35%, in HSCT cohort)	NA	0 (Median follow-up of 18 mo)	NA	3-year RFS: 52% vs. 48% (Among CR/CRi pts, with or without censoring HSCT/other subsequent therapy)	NA
John et al. [249]	Prospective (Real world data from CIBMTR)	13.8 (Range: 0.4–25.9)	CD19-4-1BBz (tisa-cel)	96 (19.3%, 96/497)	CR/CRi	195 (25.4%, all cohort)	NA	NA	NA	Median RFS: 36.02 mo vs. 36.02 mo (Among<18-year-old pts with or without censoring HSCT); Median RFS: 14.7 mo vs. 15.76 mo (Among ≥ 18-year-old pts with or without censoring HSCT); Median OS: 29.54 mo vs. 26.22 mo (Among ≥ 18-year-old pts with or without censoring HSCT);	NA
Roddie et al. [3]	Prospective (FELIX)	47 (Range: 20–81)	CD19-4-1BBz (obe-cel)	18 (18%, 18/99)	CR/CRi	6 (33%, in HSCT cohort)	NA	NA	NA	No substantial difference in EFS or OS between HSCT and non-HSCT cohorts	NA
Wang et al. [4]	Prospective	32 (Range: 18–58)	CD19-4-1BBz (inati-cel)	12 (29%, 12/41)	CR/CRi	8 (16.7%, all cohort)	2 (16.7%)	NA	NA	Median DOR: 20.7 mo vs. 20.7 mo (with or without censoring HSCT)	NA
An et al. [51]	Prospective	22 (Range: 3–72)	CD19-4-1BBz	10 (26.3%, 10/38)	CR/CRi	9 (19.1%, all cohort)	NA	NA	NA	Time-dependent Cox model shows that HSCT is not significantly associated with improved RFS (HR: 0.533, P = 0.285) and OS (HR: 0.187, P = 0.105)	NA
Aldoss et al. [32]	Prospective	38 (Range: 22–72)	CD19-CD28z	21 (52.5%, 21/40)	CR/CRi	10 (47.6%, in HSCT cohort)	1 (4.8%)	2 (9.5%)	NA	Relapse: 2 (9.5%) vs. 11 (57.9%); Multivariable Cox analysis shows that HSCT is associated with improved RFS (adjusted HR: 0.16, P = 0.001)	NA
Gardner et al. [62]	Prospective	12.3 (Range: 1.3–25.4)	CD19-4-1BBz	11 (27.5%, 11/40)	MRD-CR	28 (62%, all cohort)	NA	2 (18.1%)	NA	Relapse: 2 (18.1%) vs. 16 (55.2%)	NA
Pan et al. [250]	Prospective	11 and 24 (Two cohorts, range:2–68)	CD19-4-1BBz	27 (60%, 27/45)	CR/CRi	NA	2 (7.4%)	2 (7.4%, with a median follow-up of 133 days)	6-month LFS: 81.3%	Relapse: 2 (7.4%) vs. 9 (50%)	NA
Frey et al. [251]	Prospective	33.8 (Range: 20.6–70.4)	CD19-4-1BBz (tisa-cel)	9 (37.5%, 9/24)	CR	13 (37%, all cohort)	NA	NA	NA	Landmark analysis shows that HSCT is associated with improved EFS (P = 0.029), but OS improvement is not significant (P = 0.09)	NA
Shah et al. [252]	Prospective	13.5 (Range: 4.3–30.4)	CD19-CD28z	21 (75%, 21/28)	MRD-CR	4 (19%, in HSCT cohort)	6 (28.6%)	2 (9.5%)	24-month CIR: 9.5%; Median OS: 70.2 mo; 5-year EFS: 61.9%	Relapse: 2 (9.5%) vs. 7 (100%)	NA
Wayne et al. [253]	Prospective	13.5 (Range: 3–20)	CD19-CD28z (brexu-cel)	14 (87.5%, 14/16)	CR/CRi (n = 14); Unknown (n = 2)	5 (31.3%, in HSCT cohort)	NA	NA	Median RFS: 9.1 mo; Median DOR: 10.7 mo (in 14 CR/CRi pts)	Median DOR: 7.2 mo vs. 14.2 mo (with or without censoring HSCT)	NA
Hay et al. [254]	Prospective	39 (Range: 20–76)	CD19-4-1BBz	18 (40%, 18/45)	MRD-CR	23 (43%, all cohort)	4 (23%)	3 (17%, with a median follow-up of 28.4 mo)	2-year CIR: 17%; 2-year EFS: 61%; 2-year OS: 72%	Relapse: 3 (17%) vs. 19 (70%); Multivariable analysis shows that HSCT is associated with improved EFS, but not significant (HR: 0.39, P = 0.088)	NA
Jiang et al. [247]	Prospective	28 (Range: 10–65)	CD19-4-1BBz	21 (44.7%, 21/47)	MRD-CR	3 (5.2%, all cohort)	2 (9.5%)	2 (9.5%)	NA	Relapse: 2 (9.5%) vs. 16 (61.5%); HSCT cohort exhibits superior EFS (P = 0.021), and RFS (P = 0.001); Multivariate Cox regression analysis shows that HSCT is associated with improved EFS (HR: 0.123, P = 0.001) and RFS (HR: 0.065, P = 0.001)	For pts with high pre-infusion MRD (≥ 5%), HSCT is associated with improved EFS (P = 0.032) and RFS (P = 0.0031); For pts with low pre-infusion MRD (<5%) and poor prognostic cytogenetic markers, HSCT is associated with improved EFS (P = 0.03) and RFS (P = 0.061); For pts with low pre-infusion MRD (<5%) but without poor prognostic cytogenetic markers, no significant differences in EFS, RFS and OS among HSCT and non-HSCT cohorts
Zhang et al. [246]	Retrospective	NA (Range: 1–75)	CD19-CD28z/CD19-4-1BBz	184 (87.2%, 184/211)	CR	NA	NA	32 (17.4%)	2-year LFS: 60.4%; 2-year OS: 68.1%	Relapse: 32 (17.4%) vs. 11 (40.7%); 2-year LFS: 60.4% vs. 27.8%, P<0.0001; 2-year OS: 68.1% vs. 28.3%, P<0.0001; Multivariable analysis shows that HSCT is associated with improved LFS (HR: 2.7, P<0.001) and OS (HR: 2.5, P<0.001)	For HSCT cohort, no significant differences in 2-year LFS (P = 0.695) and 2-year OS (P = 0.608) among different BM burden groups; For BM burden<5% pts, HSCT is associated with improved 2-year LFS (65.7% vs. 46.7%, P = 0.014) and 2-year OS (69.1% vs. 40%, P = 0.005); For TP53 + pts, HSCT is associated with improved 1-year LFS (58.8% vs. 16.7%, P = 0.0037) and 1-year OS (69.5% vs. 16.7%, P = 0.0021)
Zhao et al. [255]	Retrospective	27 (Range: 3–65)	CD19-4-1BBz	55 (45.1%, 55/122)	MRD-CR (n = 40); MRD + CR (n = 15)	3 (5.5%, in HSCT cohort)	2-year NRM: 6.8%	11 (20%, with a median follow-up of 613 days)	2-year LFS: 65.6%; 2-year OS: 77.0%	Relapse: 11 (20%) vs. 44 (65.7%); 2-year CIR: 27.6% vs. 67.2%, P<0.001; 2-year LFS: 65.6% vs. 32.8%, P<0.001; 2-year OS: 77.0% vs. 36.4%, P<0.001	For HSCT cohort, age ≥ 40 and MRD+ before HSCT are associated with inferior LFS (Age: HR: 4.71, P = 0.004; MRD: HR: 4.47, P = 0.005) and OS (Age: HR: 7.11, P = 0.001; MRD: HR: 3.7, P = 0.035)
Li et al. [256]	Retrospective	13 (Range:1–57)	CD19-4-1BBz/CD19-CD22-4-1BBz	17 (47.2%, 17/36)	CR	NA	NA	0	NA	Relapse: 0 vs. 7 (36.8%); HSCT cohort exhibits superior EFS (P = 0.002) and OS (P = 0.008); Multivariate Cox regression analysis shows that HSCT is associated with improved EFS (HR: 0.247, P = 0.008) and OS (HR: 0.279, P = 0.014)	NA
Summers et al. [245]	Retrospective	12 (Range: 1–22, in HSCT cohort)	CD19-4-1BBz	23 (46%, 23/50)	MRD-CR	10 (43%, in HSCT cohort)	1 (4.3%)	5 (21.7%)	2-year LFS: 74%	Relapse: 5 (21.7%) vs. 19 (70.4%); Univariate analysis shows that HSCT is associated with improved LFS (HR: 0.31, P = 0.01)	For HSCT naïve pts, HSCT is associated with improved LFS, but not significantly (HR: 0.11, P = 0.09); For early BCA (≤ 63 days) pts, HSCT is associated with improved LFS (HR: 0.16, P = 0.01)
Gu et al. [244]	Retrospective	NA (Range: 16–60)	CD19-4-1BBz	30 (58.8%, 30/51)	MRD-CR/CRi (n = 22); MRD + CR/CRi (n = 8)	NA	2-year TRM: 14.3%	7 (23.3%)	2-year LFS: 53.2%; 2-year OS: 58.9%;	2-year CIR: 36.9% vs. 78.4%, P<0.001; 2-year LFS: 53.2% vs. 18.8%, P<0.001; 2-year OS: 58.9% vs. 22.7%, P = 0.005; Multivariable analysis shows that HSCT is associated with improved both LFS (HR: 0.2, P<0.001) and OS (HR: 0.29, P = 0.001)	For HSCT cohort, MRD- pts exhibited superior LFS (P = 0.001) and OS (P = 0.022) than MRD + pts; For MRD- cohort, HSCT is associated with improved LFS (P = 0.001) and OS (P = 0.027); For MRD+ cohort, HSCT is associated with improved LFS (P = 0.008) and OS (P = 0.014);
Roloff et al. [257]	Retrospective (Real world data from ROCCA)	46 (Range: 18–81)	CD19-CD28z (brexu-cel)	30 (19.9%, 30/151)	MRD-CR (n = 26); Unknown (n = 4)	4 (13.3%, in HSCT cohort)	NA	NA	12-month PFS: 74%; 12-month OS: 77% (among 26 MRD-CR pts)	Multivariable analysis shows that HSCT is associated with improved PFS (HR: 0.34, P = 0.02)	NA
Lust et al. [42]	Retrospective (Real world data from PRWCC and ROCCA)	22.5 (brexu-cel) and 21 (tisa-cel) (Range: 18–26)	CD19-CD28z (brexu-cel)/ CD19-4-1BBz (tisa-cel)	11 (18.3%, 11/60)	CR	23 (32.9%, all cohort)	NA	NA	NA	Multivariable analysis shows that HSCT has no significant impacts on DOR, (HR: 0.43, P = 0.2) RFS (HR: 0.47, P = 0.15) and OS (HR: 0.54, P = 0.38)	NA

Allo-HSCT: Allogeneic-hematopoietic stem cell transplantation; CR: Complete remission; CIR: Cumulative incidence of relapse; CRISPR: Clustered Regularly interspaced short palindromic repeats; CRS: Cytokine release syndrome; Cy: Cyclophosphamide; DL: Dose level; DOR: Duration of remission; EBV: Epstein-Barr virus; EFS: Event free survival; Flu: Fludarabine; HLA: Human leukocyte antigen; HR: Hazard ratio; LD: Lymphodepletion; MRD: Minimal residual disease; NA: Not applicable; NRM: Non-relapse mortality; PFS: Progression free survival; OS: Overall survival; RFS: Relapse free survival; sCRS: Severe cytokine release syndrome; sICANS: Severe immune effector cell associated neurotoxicity syndrome; TRM: Transplant-related mortality

Beyond relapse risk factors, other elements influence allo‑HSCT decisions. In the ELIANA trial, pediatric/young adult patients who achieved CR with tisa-cel and did not receive allo-HSCT had 2-year and 3-year RFS rates of 81% and 76%, respectively, whereas those who received transplant therapy or other consolidation had a 3-year RFS rate of 48% (52% when censoring for consolidation) [248]. Real‑world CIBMTR data confirmed that pediatric patients can achieve durable remission with CAR‑T therapy alone (median RFS of 36 months) [249], suggesting that the use of CAR‑T as a definitive therapy is feasible in this population. In contrast, most studies indicate that adult patients benefit from post‑CAR‑T allo‑HSCT [32, 251, 254, 257], leading to the recommendation for consolidative allo-HSCT in adult B-ALL patients who achieve remission after CAR-T therapy [258].

The effect of the CAR‑T costimulatory domain on transplant decisions remains unclear. In the ZUMA-4 study (children/young adults treated with brexu-cel), most patients received allo-HSCT; the median DOR censored for transplant was 7.2 months (vs. 14.2 months uncensored), and the median RFS censored was 5.2 months (vs. 7.4 months) [253]. Shah et al. [252] reported that among adults who achieved CR with CD28-based CAR-T therapy, all 7 nontransplanted patients relapsed, whereas only 2 of the 21 transplanted patients relapsed (5-year EFS of 61.9%), suggesting that CD28-based CAR-T may require transplantation to compensate for their limited persistence. Whether patients with prior allo‑HSCT benefit from a second transplant after receiving CAR‑T is debated, with one study showing no difference in 2‑year LFS or OS between the first and second transplants [259]. Donor selection, time intervals, and conditioning regimens may also affect transplantation outcomes. Future large-scale randomized controlled trials are warranted to evaluate the role of consolidative allo-HSCT, along with more subgroup-specific research to provide personalized clinical decision-making for B-ALL patients.

Beyond allo-HSCT, other consolidation strategies have been explored to increase CAR‑T persistence. Insufficient CD19 antigen exposure is associated with poor persistence of CD19 CAR‑T in vivo. To address this problem, combining CD19 CAR‑T with autologous T cells artificially expressing CD19 (T‑APCs) provides intermittent antigen stimulation to increase CAR‑T proliferation and persistence. In the PLAT-03 trial (19 R/R B-ALL patients), T-APC infusion caused no CRS or neurotoxicity and significantly prolonged CAR-T persistence (P = 0.03) [260]. Another study in 13 newly diagnosed Ph + B‑ALL patients used multicycle CD19 CAR‑T, CD19‑expressing T cells, and TKIs for consolidation therapy, achieving a median DOR of 27 months, with RFS and OS rates of 84% and 83%, respectively [261]. Dendritic cell (DC) vaccines can also promote CAR‑T expansion. In a study of 8 R/R B-ALL patients who achieved MRD-negative CR after receiving CD19 CAR‑T, subsequent DC vaccination induced CAR‑T re‑expansion with a median persistence of 336 days, outperforming historical CAR‑T monotherapy [262]. These findings suggest that such consolidation strategies may offer alternatives to allo-HSCT in B-ALL patients. Nevertheless, these innovative approaches require further validation in larger studies.

Salvage Therapy

Although CD19 CAR‑T cells achieve high remission rates in patients with R/R B‑ALL, approximately 50% of patients relapse after CAR‑T infusion, and a subset fail to achieve remission after the first infusion. On the basis of CD19 expression on leukemic blasts, refractory or relapsed disease can be classified as antigen‑positive or antigen‑negative, guiding the selection of distinct salvage strategies.

Blasts from CD19-negative patients often retain other B‑cell antigens, making alternative antigen targeting a key salvage strategy. CD22 is the most commonly used target. InO can be used after CD19-negative relapse [263], and CD22 CAR‑T therapy induces remission in approximately 60%–80% of such patients, although the responses are often limited by CD22 downregulation [264–267]. Therefore, bridging to HSCT after CD22 CAR-T is recommended to consolidate responses. Additionally, several preclinical strategies aimed at improving CAR-T sensitivity to antigen recognition may help enhance the clearance of target cells with low antigen density [268–270], although the efficacy of these strategies requires clinical validation.

Lineage switching, a special form of antigen‑negative relapse, involves the loss of lymphoid antigens, which renders alternative lymphoid antigen targeting ineffective. No consensus exists on salvage therapy for lineage switching. Case reports have described successful reinduction with gemtuzumab ozogamicin plus fludarabine and high-dose cytarabine [271] or with dasatinib and venetoclax in a BCR::ABL1 + patient [272].

For CD19-positive disease, the reinfusion of CD19 CAR-T cells can be used as salvage therapy. However, compared with initial CAR-T therapy, reinfusion induces lower CR rates, with approximately 20–50% of B-ALL patients achieving a second remission after reinfusion of the same CAR-T product [273–275]. A potential reason is that repeated infusions of murine-derived CAR-T cells can induce anti-CAR immune responses in patients. Humanized CAR‑T cells with lower immunogenicity have achieved improved outcomes: one study reported a CR/CRi rate of 79% (26/33), with a median RFS of 29 months [276]. Alternatively, InO or blinatumomab can be used, offering greater accessibility without manufacturing delay than CAR-T-cell reinfusion does. Wudhikarn et al. [277] used blinatumomab ± InO to treat 7 B-ALL patients with post-CAR-T-cell antigen-positive relapse, of whom 3 (43%) achieved CR. Qi et al. [278] used blinatumomab as salvage therapy in 5 B-ALL patients who had refractory disease or who had relapsed after receiving CD19 CAR-T therapy, and 4 (80%) patients achieved CR/CRi. A recent case report indicated that donor-derived CAR‑NK therapy salvaged a CAR‑T‑refractory pediatric B‑ALL patient, inducing remission and enabling HSCT [279]. Given the small sample sizes of existing studies, more research is warranted to evaluate the efficacy and safety of these salvage strategies in patients with CD19-positive disease.

Moving CAR-T therapy to earlier lines of B-ALL treatment

Given the potent antileukemic activity of CAR‑T in R/R B‑ALL, moving CAR-T to earlier lines of treatment is an important trend. Currently, patients receiving CAR‑T have typically undergone multiple prior therapies. Extensive prior treatment compromises bone marrow reserve and T-cell quality, correlating with poor outcomes [22, 225, 231]. Early application (e.g., at first relapse or initial diagnosis) could reduce cumulative chemotherapy toxicity, simplify treatment, and improve long-term prognosis, especially for high-risk patients. Using CAR‑T at lower disease burdens—such as post-remission consolidation or MRD eradication—also mitigates CRS/ICANS risk, enables deeper clearance, and may reduce subsequent therapy needs (Table 5).

Table 5.

Early use of CAR-T for B-ALL patients

Reference	CAR-T design	Number of patients receiving CAR-T infusion	Median age	Timing of CAR-T therapy	CR/CRi rate	CRS/sCRS	ICANS/sICANS	Long term outcomes	Number of patients bridging to transplantation
Zhang et al. [203]	Sequential CD19/CD22 CAR-T	27(including 25 pts receiving CD22 CAR-T)	48.5 (Range: 18–69)	First-line consolidation (after vindesine/dexamethasone/dasatinib induction)	85%CMR (23/27, after CD19 CAR-T); 76%CMR (19/25, after CD22 CAR-T)	21/0	0	2-year LFS: 92%; 2-year OS: 92%	1
Li et al. [218]	Tandem CD19/CD22 CAR-T	12	38 (Range: 18–57)	First-line therapy (after venetoclax/azacitidine induction)	91.7% (11/12, 10 MRD negative)	8/1	0	1-year CIR: 18.2; 1-year LFS: 81.8%; 1-year OS: 81.8%	10
Niu et al. [183]	Tandem CD19/CD22 CAR-T	15	51 (Range: 23–70)	First-line consolidation (MRD positive after induction, n = 11); MRD relapse or MRD positive remission after salvage therapy (n = 4)	100% (15/15, 15 MRD negative)	4/0	1/0	12-month RFS: 77%; 12-month OS: 86%	3
Chen et al. [261]	CD19 CAR-T	13	41 (Range: 27–58)	First-line consolidation (Combined with TKI and CD19 engineered T cells)	-	9/0	0	Median DOR: 27 mo; Median follow-up: 27 mo; RFS: 84%; OS: 83%	0
Schwartz et al. [280]	CD19 CAR-T	7	48 (Range: 18–69)	First-line consolidation (MRD positive after induction)	85.7% (6/7, 6 MRD negative)	0	0	6 pts remained in NGS-MRD negative remission at a median of 3 mo (Range: 1–5 mo) post CAR-T	NA
Aldoss et al. [281]	Memory-enriched CD19 CAR-T	13	66 (Range: 55–79)	First-line consolidation (Regardless of MRD)	-	7/0	0	With a median follow up of 175 days (Range: 44–443), 10 pts remained in MRD negative remission	0

Allo-HSCT: Allogeneic-hematopoietic stem cell transplantation; CR: Complete remission; CIR: Cumulative incidence of relapse; CMR: Molecular complete remission; DOR: Duration of remission; EFS: Event free survival; MRD: Minimal residual disease; NA: Not applicable; NGS: Next generation sequencing; NRM: Non-relapse mortality; PFS: Progression free survival; OS: Overall survival; RFS: Relapse free survival; sCRS: Severe cytokine release syndrome; sICANS: Severe immune effector cell associated neurotoxicity syndrome; TKI: Tyrosine kinase inhibitor

CAR‑T cells have been explored as frontline therapy for newly diagnosed B‑ALL. Zhang et al. [203] treated newly diagnosed Ph + B‑ALL adults with dasatinib‑based induction followed by sequential CD19/CD22 CAR‑T therapy. Among the 27 patients, 85% achieved molecular CR after receiving CD19 CAR‑T, and 76% of the 25 patients who received CD22 CAR-T also achieved molecular CR. At a median follow‑up of 23.9 months, only two relapses occurred, and the 2-year LFS and OS were both 92%. Similarly, Li et al. [218] used venetoclax/azacitidine induction followed by tandem CD19/CD22 CAR‑T cells in 12 newly diagnosed high‑risk Ph‑negative B‑ALL patients. The CR rate was 91.7% (10 MRD‑negative). After a median follow‑up of 17 months, three patients relapsed (all CD19‑positive), and in 1-year CIR, LFS, and OS of 18.2%, 81.8%, and 81.8%, respectively.

CAR‑T as consolidation therapy for MRD‑positive B‑ALL have also been evaluated in several studies. Niu et al. [183] treated 15 adult patients (11 as first‑line consolidation, 4 after salvage for relapse); all achieved MRD‑negative remission. Among the 11 first‑line patients, only one received allo‑HSCT, and the 12‑month RFS and OS for the remaining non‑transplanted patients were 77.8% and 80.8%, respectively. Schwartz et al. [280] infused CD19 CAR‑T in 7 first‑remitted MRD‑positive adults; 6 achieved NGS‑MRD negativity at a median of 3 months. Additionally, in the study by Aldoss et al., [281] older (≥ 55 years) B-ALL patients in first remission, regardless of their MRD status, received CAR-T as final consolidation therapy. Among the 11 enrolled patients, the treatment was well tolerated. After a median follow-up of 175 days, only one patient with Ph + B-ALL experienced molecular relapse, while the remaining patients remained in MRD-negative remission. These preliminary data suggest that the use of CAR-T cells as post-remission consolidation therapy may maintain deep remission in high-risk or MRD-positive patients, potentially offering an alternative to transplantation.

In conclusion, the early use of CAR‑T in B‑ALL patients, especially for high‑risk patients, is an attractive strategy but requires further validation. Key unresolved issues include the following: the optimal sequence of CAR‑T with other immunotherapies (e.g., blinatumomab and inotuzumab) that are increasingly used on the front line; [282] whether CAR‑T consolidation can reduce subsequent treatment or prevent CNS relapse; and the high cost limiting widespread early application.

Conclusion

CAR-T therapy constitutes a milestone in the treatment of R/R B-ALL, achieving remarkable CR rates and offering a potent therapeutic option for patients who have exhausted conventional therapies. Through the continuous optimization of CAR design, manufacturing processes, and combination strategies, CAR-T products have evolved to enhance efficacy, reduce toxicity, and improve accessibility.

Nevertheless, several challenges impede long-term success, including antigen escape relapse, limited CAR-T-cell persistence, and significant toxicity effects, such as CRS, ICANS, and hematotoxicity. Promising strategies under investigation to address these limitations include dual-targeting CARs, CRISPR-engineered CAR-T, allogeneic CAR-T products, and rational combinations with other agents. Furthermore, integrating CAR-T therapy into earlier lines of treatment, including first-line or first-relapse settings, is an important future direction to maximize curative potential while mitigating cumulative toxicities associated with multiple lines of therapy.

In conclusion, while CAR-T therapy has transformed the therapeutic landscape for B-ALL, its full potential is still evolving. Future efforts should focus on validating novel approaches to overcome resistance and poor persistence, improving safety profiles, optimizing treatment timing, and broadening accessibility to fully realize the curative promise of CAR-T therapy.

Abbreviations

allo-HSCT

Allogeneic-hematopoietic stem cell transplantation

ASTCT

American Society for Transplantation and Cellular Therapy

AUC

Area under the curve

B2M

β2-microglobulin

B-ALL

Acute B-lymphoblastic leukemia

BCA

B cell aplasia

BCR

B cell recovery

BTKi

Bruton’s tyrosine kinase inhibitors

CAR-T

Chimeric antigen receptor T cell

Cas9

CRISPR-associated protein 9

CIR

Cumulative incidence of relapse

CNS

Central nervous system

CR

Complete remission

CRi

Complete remission with incomplete hematologic recovery

CRISPR

Clustered regularly interspaced short palindromic repeats

CRS

Cytokine release syndrome

CTLA-4

Cytotoxic T-lymphocyte-associated protein 4

Cy

Cyclophosphamide

DC

Dendritic cell

DOR

Duration of response

EFS

Event free survival

EMD

Extramedullary disease

F-CAR-T

Fast CAR-T

Flu

Fludarabine

G-CSF

Granulocyte colony-stimulating factor

GvHD

Graft-versus-host disease

HDACi

Histone deacetylase inhibitors

HG

Hypogammaglobulinemia

HLA

Human leukocyte antigen

HLH

Hemophagocytic lymphohistiocytosis

ICAHT

IMMUNE Effector Cell-Associated Hematotoxicity

ICANS

Immune effector cell associated neurotoxicity syndrome

IEC-HS

Immune effector cell-associated hemophagocytic lymphohistiocytosis

IL

Interleukin

iNKT

Invariant Natural Killer T

iPSCs

Induced pluripotent stem cells

IVIG

Intravenous immunoglobulin

KI

Knock-in

LD

Lymphodepletion

LFS

Leukemia-free survival

MHC

Major histocompatibility complex

MM

Multiple myeloma

MRD

Minimal residual disease

NfL

Neurofilament light chain

NGS

Next generation sequencing

ORR

Objective response rate

OS

Overall survival

PD-1

Programmed cell death protein 1

PFS

Progression-free survival

POC

Point-of-care

PR

Partial remission

RFS

Relapse-free survival

R/R

Relapsed/refractory

scFv

single-chain variable fragment

TCR

T cell receptor

TIM-3

T cell immunoglobulin and mucin domain-containing protein 3

TKI

Tyrosine kinase inhibitor

Tregs

Regulatory T cells

TRUCK

T cells Redirected for Universal Cytokine-mediated Killing

UCB

Umbilical cord blood

Author contributions

CZ: Writing original draft, Visualization. YW: Review and editing, Funding acquisition.

Funding

This work was supported by CAMS Innovation Fund for Medical Sciences (2024-I2M-C&T-B-082), CAMS Innovation Fund for translation (2024-I2M-ZH-015), Tianjin key medical discipline construction project (No.TJYXZDXK-3–001 A).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

Not applicable.

Consent to participate and publish

Not applicable.

Competing interests

The authors declare no competing interests.