Purpose:

The survival rate of children with refractory/relapsed acute myeloid leukemia (R/R-AML) by salvage chemotherapy is minimal. Treatment with chimeric antigen receptor T cells (CAR T) has emerged as a novel therapy to improve malignancies treatment. C-type lectin-like molecule 1 (CLL1) is highly expressed on AML stem cells, blast cells, and monocytes, but not on normal hematopoietic stem cells, indicating the therapeutic potential of anti-CLL1 CAR T in AML treatment. This study aimed to test the safety and efficacy of CAR T-cell therapy in R/R-AML.

Patients and Methods:

Four pediatric patients with R/R-AML were enrolled in the ongoing phase I/II anti-CLL1 CAR T-cell therapy trial. The CAR design was based on an apoptosis-inducing gene, FKBP-caspase 9, to establish a safer CAR (4SCAR) application. Anti-CLL1 CAR was transduced into peripheral blood mononuclear cells of the patients via lentivector 4SCAR, followed by infusion into the recipients after lymphodepletion chemotherapy. Cytokine release syndrome, immune effector cell–associated neurotoxicity syndrome, and other adverse events were documented. Treatment response was evaluated by morphology and flow cytometry–based minimal residual disease assays.

Results:

Three patients with R/R-AML achieved complete remission and minimal residual disease negativity, while the other patient remained alive for 5 months. All these patients experienced low-grade and manageable adverse events.

Conclusions:

On the basis of our single-institution experience, autologous anti-CLL1 CAR T-cell therapy has the potential to be a safe and efficient alternative treatment for children with R/R-AML, and therefore requires further investigation.

Translational Relevance

The outcomes of children with refractory/relapsed acute myeloid leukemia (R/R-AML) are very disappointing. The prognosis for patients with R/R-AML can be greatly enhanced if they are in a negative minimal residual disease (MRD) state even if they are not subsequently treated with allo-HSCT, thus novel bridging therapies are urgently needed. In this study, we enrolled four children with R/R-AML for a phase 1/2 trial with autologous anti-CLL1-based CAR-T cell therapy to test its safety and efficacy. Three patients achieved complete remission with MRD negativity, and the other patient remained stable for five months. All patients experienced low-grade but manageable adverse events. To this end, our study adds clinical value to the safety and efficacy CAR-T cells in AML therapy, thus we believe that CAR-T cells therapy would be a promising therapeutic option for R/R-AML.

Approximately 50% to 70% of the children with acute myeloid leukemia (AML) can be effectively treated with conventional therapy (1). The 5-year survival rate of childhood AML varies between 33.3% and 79.5% worldwide, as reported by the CONCORD-2 study (2). However, pediatric patients with relapsed and/or refractory AML (R/R-AML) respond poorly to the salvage chemotherapy currently available, and novel therapies are warranted to improve the treatment outcome (3). Currently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is accepted as the standard treatment for R/R-AML and it has been reported that outcomes for patients with R/R-AML were enhanced in patients with negative minimal residual disease (MRD) state prior to allo-HSCT (4). Therefore, novel bridging therapies are urgently needed.

Autologous CAR T-cell therapy has emerged as a highly effective regimen for relapsed/refractory hematologic malignancies (5, 6); however, its efficacy in AML remains unclear. Recent research has shown that CLL1 is highly expressed on AML stem cells and blast cells but not on normal HSCs (7, 8), suggesting that CLL1 is a promising target for novel AML therapy that might not impair normal hematopoiesis. Intriguingly, several groups have developed novel CLL1-directed therapies with definite efficacy on AML cell lines, primary human AML cells, and human AML patient-derived xenograft mice and monkey models (9). Two recent reports have claimed success of CLL1-based CAR T cells in patients with secondary AML (10, 11), thereby highlighting its potential in R/R-AML. However, direct evidence of CAR T-cell treatment response from patients with R/R-AML remains limited.

Here we reported primary findings on the effects of autologous CLL1-based CAR T-cell therapy in four children with R/R-AML.

Eligibility, ethics approval, and treatment schema

This study was approved by the Institutional Review Board of Guangzhou Women and Children's Medical Center (Guangdong, P.R. China; 2018050201, 2018050202, 2018050803, and 2020-23) and Shenzhen Geno-immune Medical Institute, Shenzhen, P.R. China (GIMI-IRB-16001). This clinical trial was registered at www.clinicaltrials.gov (No. NCT03222674). Pediatric patients with AML were initially enrolled in the CALSIII-AML18 trial (ChiCTR1800015883). If they were refractory to CALSIII-AML18 chemotherapy or relapsed after complete remission (CR), they were classified as R/R-AML and were considered eligible for the anti-CLL1 CAR T-cell therapy trial. Additional inclusion criteria included the following: (i) age older than 2 years; (ii) identification of CD33, CD38, CD56, CD123, MucI, and CLL1 expression in malignant cells by IHC staining or flow cytometry; (iii) a Karnofsky performance status score higher than 80 and life expectancy >2 months; (iv) adequate bone marrow (BM), liver, and renal function as assessed by cardiac ejection fraction ≥50%, oxygen saturation ≥90%, creatinine ≤2.5× upper limit of normal, aspartate aminotransferase and alanine aminotransferase ≤3 × upper limit of the normal levels, total bilirubin ≤2.0 mg/dL; (v) Hgb ≥80 g/L; (vi) ease of obtainment of T cells for CAR T-cell preparation; and (vii) the ability to understand and the willingness to provide written informed consent by the parents. Patients were excluded from the study if they met any of the following criteria: (i) a severe illness or medical condition, which would not permit the management of the patient according to the protocol; (ii) presence of active bacterial, fungal, or viral infection not controlled by adequate treatment; (iii) history of glucocorticoid therapy within the week prior to entering the test; (iv) previous treatment with any gene therapy products; and (v) patients who, based on the investigator's opinion/judgment, would not be able to comply with study's procedures and requirements. Informed consent was obtained from the parents or guardians, according to the institutional guidelines and the Declaration of Helsinki.

Study design

All patients enrolled in this trial received one lymphodepletion treatment cycle according to the cyclophosphamide plus fludarabine regimen proposed earlier (cyclophosphamide, 900 mg/m2/day, day −4 to day −1; fludarabine, 30 mg/m2/day, varied from day −4 to day −1; ref. 10), before CAR T cell transfer to enhance in vivo expansion of CAR T cells, after which the patients received a single dose (a dose of at least 1 × 106 anti-CLL1 CAR T cells/kg) infusion over 10 minutes through a peripherally inserted central venous catheter. The treatment response and CAR T-cell–related toxicities were documented as described below. BM morphologic and flow cytometric assessments (the sensitivity is 10–3) for treatment response were performed every month for the first 3 months after CAR T-cell therapy, and every 3 months thereafter. During the therapy, pneumocystis prophylaxis (trimethoprim/sulfamethoxazole or dapsone) and antifungal prophylaxis (posaconazole) were prescribed until the white blood cells (WBCs) counts recovered over the required limit of 0.5 × 109/L. No prophylactic antibiotics were required. Patients achieving CR received allo-HSCT if their socioeconomic conditions allowed them to afford further therapy.

Construction of the 4SCAR-CLL1 lentiviral vector and production of clinical-grade CLL1 CAR T cells

Lentiviral vectors (LVs) were generated on the basis of the NHP/TYF LV system, as per previously described methods (12, 13). A new fourth-generation CAR, containing CLL1-specific single-chain variable fragments, derived from a humanized mAb based on patent WO2017091615A1, which was fused with CD28-CD27-CD3z signaling domains and an inducible caspase 9 motif, was chemically synthesized and cloned into the pTYF transducing vector downstream a human EF1α promoter (14–16). The sequence and functionality of the final, developed LV-CAR construct was extensively verified by functional analyses. 4SCAR-CLL-1 target specific analysis was comprehensively conducted in preclinical studies using AML patient-derived CLL-1 CAR T cells and CLL-1–positive AML cell lines, THP-1 and U937 (data not shown).

Peripheral blood mononuclear cells (PBMCs) were obtained from the patients via apheresis. Lentiviral CLL1-CAR–modified T cells were generated from fresh PBMCs as per previously described methods (10). Briefly, CD3+ T cells were isolated and activated using anti-CD3/CD28 beads at a 1:1 bead-to-cell ratio (Miltenyi Biotec) in AIM-V (Thermo Fisher Scientific) supplemented with 5% human AB serum (Valley Biomedical), 2 mmol/L Glutamax (Thermo Fisher Scientific), and IL2, IL7, and IL15 (PeproTech) as per previously described protocols (17). Cells were transduced on days 2 or 3 with the 4SCAR-CLL1 LVs and expanded for 4 days in media containing IL2, IL7, and IL15. The 4SCAR-CLL1 LV gene transfer efficiencies for PBMCs of patients varied from 13.54% to 95.05%. All protocols were used under GLP cell manufacture and GMP viral vector production practice following the regulatory guidelines.

CAR detection by quantitative PCR

Genomic DNA was extracted from blood cells using the Promega Genomic DNA Purification Kit (Promega Corp.). The CAR copy number in blood was determined by quantitative real-time PCR (qPCR) based on the SYBR and Taqman probe methods using the CLL1 scFv sequence-specific primer set as per previously described methods (17). The qPCR data were collected using an MX3000P (Stratagene, Agilent Technologies). To determine CAR T cell content in the peripheral blood, we used the conversion rate of CAR copies per copy of cellular genome based on internal housekeeping gene copies from the internal qPCR control. The kinetics of CLL1 CAR T cells in patients after CAR T-cell infusion was expressed as a percentage of 4SCAR-CLL1–positive cells in PBMCs based on qPCR analysis. Thus, the percentage of CAR T cells indicated the CAR T-cell content in the total PBMC population (e.g., 20% indicates 20 CAR T cells in 100 PBMCs).

Cytokine analysis based on a cytokine bead array

To analyze the levels of released cytokines in plasma, including IL2, 4, 6, and 10, TNFα, and IFNγ, we used the AimPlex BioSciences Human Premixed Multiplex (Pomona).

Examination of the treatment response

BM specimens were longitudinally collected at diagnosis, during chemotherapy, and before and after CLL1 CAR T cell infusion. The treatment response was evaluated by morphology, flow cytometry, and quantitative PCR analyses, if specific genetic alterations were present. The National Comprehensive Cancer Network (NCCN) guidelines were used for evaluating AML treatment response (18).

Safety and tolerability

The safety and tolerability of anti-CLL1 CAR T cells in the study patient population were evaluated using the Common Terminology Criteria for Adverse Events (CTCAE) 5.0 and the management guidelines for pediatric patients receiving CAR T-cell therapy (19, 20). Absolute monocyte count and the proportion of peripheral monocytes were determined at least twice a week after anti-CLL1 CAR T-cell therapy. Clinical laboratory evaluations, including complete blood cell counts, liver, and renal function, were performed when necessary according to the clinical manifestations.

Statistical analyses

Data collected were pooled for analysis, and descriptive statistics were used to summarize the data, which were analyzed using SAS Version 9.2 or higher.

Patient disposition and treatment exposure

Four children with R/R-AML (median age, 8.4 years; range, 7.3–9.6 years), were enrolled from September 2018 to March 2020, as a cross-over of our CALSIII-AML18 study. The patient characteristics and study program are shown in Table 1, Fig. 1A, and Supplementary Figs. S1 and S2. Among these four patients with R/R-AML, patient 1 was secondary AML, patient 2 presented myelodysplastic syndrome (MDS)-transformed AML and failed to respond to two cycles of induction therapy with MAG (mitoxantrone 5 mg/m2/dose, days 1–3; cytarabine 10 mg/m2/dose every 12 hours, days 1 to 10; GCSF 5 μg/kg/dose days 1–10), and patient 3 (primary AML) failed to achieve complete remission after two cycles of induction therapy and subsequent R/R-AML salvage therapy. Patient 4 experienced early relapse after HA treatment (homoharringtonine 3 mg/m2/day, days 1–5; Ara-C 3 g/m2/dose, every 12 hours, days 1–3), with 11.5% BM blasts and 13.6% MRD. All patients received one autologous anti-CLL1–based CAR T-cell dose at least 1 × 106/kg CAR T cells. A lymphodepleting chemotherapy was administered before CAR T-cell transfer to enhance the in vivo expansion of CAR T cells.

Table 1.

Clinical characteristics of these four enrolled patients with R/R-AML.

Patient No.No. 1No. 2No. 3No. 4
Age/gender 9.6/F 8.4/F 7.3/M 8.2/M 
WBC_Dx (×109/L) 2.2 66.6 10.2 
FAB subtype NOS MDS-AML M2a M6 
FISH MLLr −7 N.A. N.A. 
Karyotype 46, XX[10] 45, XX, −7[9] 46, XY[20] N.A. 
Fusion gene KMT2A-CREBBP EVI1 NUP98-NDS1 N.A. 
Mutations WT1S381fs, RUNX1R204P, TCF12P73fs NRASQ61H, NRASS65T, NRASR68T, RUNX1D198G, BRAFE501K FLT3-ITD, WT1P377fs, CEBPAQ83fs, PTPN11A72V, BRCA1I986fs, MYCP74Q, LRP2D1829E WT1R458P 
WBC pre-CAR T (×109/L) 0.9 1.1 0.4 0.7 
% blast pre-CAR T 19.5 9.5 6.5 
% MRD pre-CAR T 17.6 3.14 6.84 14.19 
CAR target CLL1 CLL1, Lewis-Y CLL1, CD33, CD38 CLL1 
Infused CAR T-cell dose (×106/kg) 1.49 1.98 1.03 
% CAR transduction efficiency 95.05 75.9 13.54 35.79 
ECOG status 
EBV/HSV/CMV/HBV/HCV status (pre-CAR T-cell therapy) No viral infection No viral infection No viral infection HSV 
EBV/HSV/CMV/HBV/HCV status (post-CAR T-cell therapy) No viral infection No viral infection CMV during HSCT HSV 
CRS grade 
ICANS grade 1 or 2 
Liver function impairment grade 
Follow-up time (months) 24 12 
MRD marker CD15 CD123 CD123 CD123 
Latest health condition (post-CAR T therapy) Died, 23 months Died, 5 months Died, 10 months Alive, 9 months 
Patient No.No. 1No. 2No. 3No. 4
Age/gender 9.6/F 8.4/F 7.3/M 8.2/M 
WBC_Dx (×109/L) 2.2 66.6 10.2 
FAB subtype NOS MDS-AML M2a M6 
FISH MLLr −7 N.A. N.A. 
Karyotype 46, XX[10] 45, XX, −7[9] 46, XY[20] N.A. 
Fusion gene KMT2A-CREBBP EVI1 NUP98-NDS1 N.A. 
Mutations WT1S381fs, RUNX1R204P, TCF12P73fs NRASQ61H, NRASS65T, NRASR68T, RUNX1D198G, BRAFE501K FLT3-ITD, WT1P377fs, CEBPAQ83fs, PTPN11A72V, BRCA1I986fs, MYCP74Q, LRP2D1829E WT1R458P 
WBC pre-CAR T (×109/L) 0.9 1.1 0.4 0.7 
% blast pre-CAR T 19.5 9.5 6.5 
% MRD pre-CAR T 17.6 3.14 6.84 14.19 
CAR target CLL1 CLL1, Lewis-Y CLL1, CD33, CD38 CLL1 
Infused CAR T-cell dose (×106/kg) 1.49 1.98 1.03 
% CAR transduction efficiency 95.05 75.9 13.54 35.79 
ECOG status 
EBV/HSV/CMV/HBV/HCV status (pre-CAR T-cell therapy) No viral infection No viral infection No viral infection HSV 
EBV/HSV/CMV/HBV/HCV status (post-CAR T-cell therapy) No viral infection No viral infection CMV during HSCT HSV 
CRS grade 
ICANS grade 1 or 2 
Liver function impairment grade 
Follow-up time (months) 24 12 
MRD marker CD15 CD123 CD123 CD123 
Latest health condition (post-CAR T therapy) Died, 23 months Died, 5 months Died, 10 months Alive, 9 months 

Abbreviations: CMV, cytomegalovirus; EBV, Epstein–Barr virus; FAB, The French-American-British classification of AML; HBV, hepatitis B; HCV, hepatitis C; HSV, herpes simplex virus; N.A., not available; NOS, not specified.

Figure 1.

CLL1 CAR T-cell therapy targeting R/R-AML. A, The treatment schema and process of patient enrolment for this study. B, The illustration of the CAR T-cell therapy schedule. C, Serum IL6 level (yellow shaded area represents the reference value). D, Body temperature monitoring during anti-CLL1 CAR T-cell treatment for R/R-AML.

Figure 1.

CLL1 CAR T-cell therapy targeting R/R-AML. A, The treatment schema and process of patient enrolment for this study. B, The illustration of the CAR T-cell therapy schedule. C, Serum IL6 level (yellow shaded area represents the reference value). D, Body temperature monitoring during anti-CLL1 CAR T-cell treatment for R/R-AML.

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Adverse events

As anti-CLL1 CAR T-cell therapy is novel strategy, it is key to evaluate its safety. Through a multidisciplinary approach based on recommendations (20), the CAR T-cell–associated toxicity, including cytokine release syndrome (CRS), immune effector cell–associated neurotoxicity syndrome (ICANS), and hemophagocytic lymphohistiocytosis (HLH) was determined. CRS and ICANS were determined at least twice a day during the first 14 days. Three patients who responded well to anti-CLL1 CAR T cells experienced grade 1 or 2 CRS (Table 1), mainly induced by IL6, manifested as fever, hypotension, and/or hypoxia, and required fluid resuscitation and/or mask oxygen inhalation (Fig. 1C and D). Other cytokine profiles were not considerably affected (Supplementary Fig. S3). In addition, ferritin levels, but not the C-reactive protein levels, were affected in a similar pattern as IL6 (Supplementary Fig. S3). CRS occurred within the first 5 days after anti-CLL1 CAR T-cell infusion and disappeared several days or weeks later (patient 1 for 15 days, patient 3 for 3 weeks, and patient 4 for 26 days; Fig. 2C and D). The longest CRS duration was registered in patients 3 and 4; however, they did not require intensive supportive care. Patient 4 had grade 1 to 2 ICANS, which manifested by transiently diminished attention, speech impediment, and tremors, but he did not experience unconsciousness or seizures, and was the only patient administered with glucocorticoids to control the CAR T-cell–related toxicities. In addition, no HLH and severe organ toxicity (grade >2) were recorded (Supplementary Table S1), suggesting a safety profile of anti-CLL1 CAR T-cell therapy. All patients experienced grade 3 anemia for the first 3 to 4 weeks of treatment. In addition, three patients (patients 1, 3, and 4) had prolonged grade 4 neutropenia for 4 to 6 weeks (Supplementary Table S2).

Figure 2.

Immunotypic features of enrolled AML cases before and after anti-CLL1 CAR T-cell treatment. The BM immunophenotypic feature of enrolled patients before and 2 weeks after anti-CLL1 CAR T-cell therapy patient 1 (A), patient 2 (B), patient 3 (C), and patient 4 (D). The BM morphologic blast percentage (E), and MRD response (F) during anti-CLL1 CAR T-cell treatment for R/R-AML (red line with solid circle, patient 1; blue line with solid square, patient 2; dark green with solid circle, patient 3; purple line with empty square, patient 4).

Figure 2.

Immunotypic features of enrolled AML cases before and after anti-CLL1 CAR T-cell treatment. The BM immunophenotypic feature of enrolled patients before and 2 weeks after anti-CLL1 CAR T-cell therapy patient 1 (A), patient 2 (B), patient 3 (C), and patient 4 (D). The BM morphologic blast percentage (E), and MRD response (F) during anti-CLL1 CAR T-cell treatment for R/R-AML (red line with solid circle, patient 1; blue line with solid square, patient 2; dark green with solid circle, patient 3; purple line with empty square, patient 4).

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Response to therapy and follow-up

In this report, the refractory AML cells well responded to anti-CLL1–based CAR T-cell therapy. The leukemic burden was quickly decreased in three patients (patients 1, 3, and 4) as assessed by flow cytometry, and CLL1 targeting was confirmed by flow cytometry (Fig. 2A–D), with a 75% overall response rate. All three responding patients (patients 1, 3, and 4) achieved morphologic leukemia-free state (Fig. 2E) 1 month after CAR T-cell therapy. In the follow-up, all these three patients achieved morphologic complete remission (CRm) and MRD-negative status as defined by flow cytometry. In terms of patient 1, the CRm and MRD status remained until 1 year after CAR T-cell therapy. Patient 1's MRD status was above 0.1% (2.21%) at 18 months after anti-CLL1 CAR T cell infusion, while her MRD status (0.37%) was not increased as evaluated at 20 months after CAR T-cell therapy (Fig. 2F; Supplementary Table S3). However, we did not observe the same outcome for patient 2, a MDS-transformed AML (MDS-tAML) case, in which, 2 weeks after CAR T-cell therapy, we detected 22.5% blasts in BM smear, and 10.92% MRD in patient 2 (Fig. 2E and F). Because of poor response, her parents declined further treatment.

The follow-up duration varied from 7 to 24 months (patient 1, 24 months; patient 2, 5 months; patient 3, 12 months; and patient 4, 8 months; Fig. 3A; Table 1). Patient 4 was still alive at the time of article submission, although he did not receive allo-HSCT due to economic reasons. Fortunately, his leukemic burden remained undetectable by microscope and flow cytometry. Regarding three other patients, we were informed that patient 1 died of persistent fever and severe bleeding 23 months after initiation of CAR T-cell therapy. In terms of patient 2, we were informed that the patient was clinically improved, with the highest hemoglobin (10.8 g/dL) concentration, and platelet (97 × 109/L), and WBC count (2.7 × 109/L) 3 months after CAR T-cell therapy. Unfortunately, patient 2 died 5 months after CAR T-cell therapy. Patient 3 received haploidentical 7/10 HLA-matched BM HSCT at 3 months after CAR T-cell therapy and had an MRD below 0.1%. This patient demonstrated complete donor chimerism 100 days after HSCT while experiencing gastrointestinal GVHD and thrombotic microangiopathy (TMA). Sadly, he died 12 months after CAR T-cell therapy due to TMA-related multiple organ dysfunction.

Figure 3.

The follow-up diagram, CLL1 targeting, and CAR T-cell expansion of patients enrolled in this anti-CLL1–based CAR T-cell therapy. A, The follow-up plot of four patients enrolled in this anti-CLL1–based CAR T-cell therapy. B, Peripheral blood absolute monocyte counts. C, CAR T-cell percentage in total PBMCs.

Figure 3.

The follow-up diagram, CLL1 targeting, and CAR T-cell expansion of patients enrolled in this anti-CLL1–based CAR T-cell therapy. A, The follow-up plot of four patients enrolled in this anti-CLL1–based CAR T-cell therapy. B, Peripheral blood absolute monocyte counts. C, CAR T-cell percentage in total PBMCs.

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CLL1 targeting and persistence of anti-CLL1 CAR T cells

The proportion of CD19-positive B cells in peripheral blood can be used to reflect the efficacy of CD19 CAR T cells. Thus, we monitored the proportion of CLL1-positive cells to evaluate the efficacy of anti-CLL1 CAR T cells. Absolute monocyte counts were found to be markedly decreased in patients 1, 3, and 4 after anti-CLL1 CAR T cell therapy, with the longest duration being almost 2 months (Fig. 2A–D; Fig. 3B). The expansion and persistence of anti-CLL1 CAR T cells were weekly determined by qPCR. Anti-CLL1 CAR T cells gradually expanded in the first week and reached its peak in the second week except in patient 4, which peaked at the third week (Fig. 3C). The expansion was then decreased to less than 1% in the second month. Surprisingly, patient 1 showed a persistently positive status for 5 months after CAR T cell infusion (Fig. 3C). Interestingly, we observed a biphasic anti-CLL1 CAR T cell expansion pattern in patient 4 (Fig. 3C); however, the exact reason could not be determined.

The goal for pediatric R/R-AML treatment is to develop new therapeutic options to achieve CR and then proceed to HSCT, thereby pursuing a cure for this disease. Three of the four patients with R/R-AML enrolled in this study benefited from the anti-CLL1 CAR T-cell therapy and presented CRm and MRD negativity, while the other patient remained alive for 5 months. Only grades 1 to 2 CRS and/or ICANS were observed and successfully managed.

The addition of novel agents to anthracyclines- and cytarabine-based chemotherapy is currently the preferred treatment for pediatric R/R-AML. Notably, several new agents benefit R/R-AML with a median 38.6% CR/complete remission incomplete (CRi) rate, while traditional chemotherapy can only achieve approximately 65.6 ± 7.0% CR/CRi3. However, novel agents may predispose patients to higher toxicity than traditional chemotherapy or could fail to sustain MRD. Moreover, patients often develop resistance after an initial response to tyrosine kinase inhibitors, such as gilteritinib (21), with treatment-related resistance as high as 37.7% (22–24).

In contrast to chemotherapy and newly FDA-approved agents, CAR T-cell therapy can minimize systemic cytotoxicity and morbidity while generating maximal anti-leukemia activity. CLL1, a novel AML-stem cell (AML-SC) marker, is abundantly expressed on AML-SC, leukemic blast cells, and monocytes but not on the normal HSCs (9); thus, it appears to be a promising target for AML therapy. Novel CLL1-directed therapies (including cell-based and antibody-based anti-CLL1 therapy) have been developed for AML treatment. For example, several researches have designed anti–CLL-1 antibody–drug conjugates (DCLL9718A, CLT030), which have been shown to remarkably target human AML cells with negligible off-target toxicity (9). Our study demonstrated that no severe organ toxicity was detected in these four patients, indicating the advantages of selective CLL1 targeting in AML therapy. Moreover, three research groups have developed and tested the efficacy of CLL-1 CAR T cells in AML using primary AML samples in vitro, and in an AML mouse model in vivo (9). These studies have demonstrated the efficiency and specificity of CLL1 CAR T cells for anti-leukemia activity. Furthermore, two previous reports have demonstrated that two secondary AML cases were successfully treated with anti-CLL1 CAR T cells (10, 11). Consistent with these findings, our data add more evidence to support CLL1 targeting in AML, albeit with a small number of cases (Figs. 13).

We observed low anti-CLL1 CAR T cell expansion in patients 1, 2, and 3. The persistence of CLL1 CAR T cells was relatively short among these four patients. The CD28/CD27 costimulatory signals could be causing the short persistence; however, a similar 4SCAR design used in trials targeting GD2 and CD19 has shown persistence of CAR T cells in neuroblastoma and B-ALL pediatric patients (25). CD28 and 4-1-BB are two well-established costimulatory signals used in the design of CAR molecules. The CD28 costimulation facilitates T-cell responses against weak agonist peptides, while 4-1-BB enhances T-cell expansion and maintenance. In this study, we observed a prolonged myelosuppression, especially the absence of monocytes, which was not attributable to chemotherapy and viral infection. Usually, the hematologic events produced by treatment with lymphodepleting chemotherapy lasted for 2 weeks, while these hematologic events lasted for 3 to 4 weeks. In addition, we could not conclude that the toxicity caused by CAR T cells was related to cytokines, as the anti-CLL1 CAR T-cell therapy could also reduce CLL1-positive normal myeloid cells including neutrophils, dendritic cells, macrophages, as well as committed myeloid progenitors. Interestingly, the absolute monocyte counts gradually recovered and CAR T cells decreased.

To date, 10 cellular therapy clinical trials have been registered at www.clinicaltrials.gov (Supplementary Table S4) for R/R-AML populations. Two trials were terminated, and five of them continued recruiting patients at the time of article submission. The candidate AML targets include FLT3, CD33, CD123, and CLL1. Most of these trials are in phase I and/or II, suggesting a considerable time lag before successful translation into the clinical management of R/R-AML. In addition to these reported targets, other AML targets, including CD7, CD13, TIM3, FLT3, and NKG2D, are reported to be effective, as evidenced by ex vivo and in vivo studies (26). Recently, CD33-CAR natural killer (NK) cells have been reported to exhibit favorable outcomes for targeting AML with no significant adverse effects (27), highlighting the potential of CAR T cells or NK cells in AML therapy.

Our clinical observation complements that of autologous anti-CLL1 CAR T-cell therapy in R/R-AML. However, here we have only reported the cases of four children with R/R-AML, which is small number of patients. Although the number of cases is small, the results demonstrated the efficacy and safety, suggesting that autologous CLL1-based CAR T-cell therapy might be an alternative option for the treatment of pediatric R/R-AML (Supplementary Fig. S4). Among these four cases, two cases did not receive allo-HSCT after remission due to economic reasons and they were still in healthy condition. Even though in this study, we have proven the efficacy of anti-CLL1 based CAR T-cell therapy, its potential for curing R/R-AML remains uncertain. Patient 3, who underwent allo-HSCT, experienced severe GVHD and TMA, however any association between anti-CLL1 CAR T-cell therapy and the development of GVHD could not be established. To summarize, further studies, conducted in a bigger cohort of patients, using the 4SCAR-CLL1 T-cell product, are necessary to further validate tolerability and efficacy of CLL1-based CAR T-cell therapy observed in the four patients enrolled in this study.

No disclosures were reported.

H. Zhang: Conceptualization, resources, formal analysis, supervision, investigation, methodology, writing–original draft, project administration, writing–review and editing. P. Wang: Data curation, writing–original draft, project administration. Z. Li: Resources, data curation, formal analysis. Y. He: Data curation, investigation. W. Gan: Conceptualization, supervision, writing–original draft, writing–review and editing. H. Jiang: Conceptualization, formal analysis, supervision, investigation, writing–review and editing.

We thank the patients and their parents for their participation, and the scientific editing from Editage (www.editage.cn) and Andy Zhao. In addition, we strongly appreciate the scientific suggestions made by Lung-ji Chang.

This work was partially funded by research funds from St. Baldrick's Foundation International Scholar (581580), the Natural Science Foundation of Guangdong Province (2015A030313460), and the Guangzhou Women and Children's Medical Center Internal Program (IP-2018-001).

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