Characteristics of anti-CLL1 based CAR-T therapy for children with relapsed or refractory acute myeloid leukemia: the multi-center efficacy and safety interim analysis

Characteristics of anti-CLL1 based CAR-T therapy for children with relapsed or refractory acute myeloid leukemia: the multi-center efficacy and safety interim analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Characteristics of anti-CLL1 based CAR-T therapy for children with relapsed or refractory acute myeloid leukemia: the multi-center efficacy and safety interim analysis Min Luo, HUI ZHANG, Chaoke Bu, Zhiyong Peng, Guangchao Li, Zhao Zhou, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1668440/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Sep, 2022 Read the published version in Leukemia → Version 1 posted 9 You are reading this latest preprint version Abstract C-type lectin like molecule-1 (CLL1) is preferentially expressed on acute myeloid leukemia (AML) stem cells and AML blasts, and can be considered as AML-associated antigen. Anti-CLL1-based CAR-T cells exhibited effective tumor killing capacity in vitro and in AML-bearing mouse model. In this report, eight children with relapsed or refractory AML (R/R-AML) were recruited for a phase 1/2 clinical trial of autologous anti-CLL1 CAR-T cell immunotherapy. The objectives of this clinical trial were to evaluate the safety and the anti-AML responses after CLL1-CAR-T cell treatment, with long-term prognosis within those patients who did not receive allogeneic hematopoietic stem cells transplantation (allo-HSCT) as an additional aim. These R/R-AML patients received one dose of autologous CLL1-CAR-T cells after lymphodepletion conditioning. Grade 3–4 hematologic adverse events were observed post CAR-T cell infusion. Meanwhile, grade 1–2 cytokine release syndrome (CRS) was observed but without any lethal events. 4 out of 8 AML patients achieved incomplete remission (CRi) and minimal residual disease (MRD) negativity, 2 patients with CRi but MRD positivity, and 2 patients with decreased AML burden and CLL1 positive AML blast clearance. These results suggested that anti-CLL1-based CAR-T cell immunotherapy can be considered as a well-tolerated and effective option for treating children with R/R-AML. relapsed refractory childhood acute myeloid leukemia CAR-T cells CLL1 Figures Figure 1 Figure 2 Introduction Outcomes of children with acute myeloid leukemia (AML) remain lagged behind that of children with acute lymphoblastic leukemia (ALL), with 5-year disease-free survival (DFS) varying between 33.3% and 79.5% worldwide, as reported by the CONCORD-2 study 1 . Though 50% – 70% of children with primary AML can be cured with conventional intensive therapy 2 , 3 , the cure rate is not improved much by the introduction of novel agents 4 . The relative poor prognosis for childhood AML has been mostly attributed to less available targeted agents or therapies for those relapsed/refractory AML (R/R-AML) patients. Up to now, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is generally considered as the best chance of cure for patients with high-risk- or R/R- AML 5 . Impressively, the prognosis for patients with R/R-AML can be greatly enhanced if they are in a negative minimal residual disease (MRD) state even without receiving allo-HSCT 6 . Therefore, novel therapies are highly needed for further improvement in the R/R-AML therapy. Several novel agents have been currently introduced into pediatric R/R-AML therapies (i.e., sorafenib, gilterinib, gemtuzumab ozogamicin (GO), and venetoclax), however the responses remain unsatisfied, with ~ 50% CR/CRi (complete remission/complete remission with incomplete hematologic recovery) rate 7 – 10 . The development of immunotherapy has given the clinicians with new strategy for treating patients with hematological malignancies. In this regard, autologous chimeric antigen receptor (CAR) T cell therapy has been increasingly accepted as a highly effective regimen for relapsed/refractory hematological malignancies 11 – 13 . For example, anti-CD19 CAR-T cell immunotherapies are reported to be highly effective in R/R B-cell ALL (B-ALL) patients, with CR rates ranging from 70 to 94% 14–16 , although near one third responding patients eventually experienced relapse due to multiple mechanisms 17 . However, the potential of CAR-T cell therapy in R/R-AML remains undetermined. C-type lectin‐like molecule‐1 (CLL1) is identified as a novel AML stem cell associated antigen 18 . Intriguingly, CLL1 is highly expressed on AML leukemia stem cells (LSCs), majority of AML blasts and normal myeloid cells, but not on normal hematopoietic stem cells (HSCs) and lymphoid cells, suggesting that targeting CLL1 can be a novel AML therapy while not affecting normal hematopoiesis and lymphocyte-directed immune function. Several studies have successfully developed novel CLL1-directed therapies (i.e., antibody-based and cellular therapies) with definite efficacy on human AML with promising ex vivo and in vivo evidences 19 – 24 . Liu and Zhang 25 have independently and successfully treated two secondary AML patients with anti-CLL1 based CAR-T cells, highlighting the potential of CAR-T cell therapy in R/R-AML. To this end, we recruited 8 children with R/R-AML for a phase 1/2 clinical trial with autologous anti-CLL1 based CAR-T cell therapy to test its safety and efficacy among two independent institutions. Materials And Methods Eligibility, ethics approval, and treatment schema This study was approved by the Institutional Review Board of Guangzhou Women and Children’s Medical Center (2020-23) and Nanfang-Chunfu Children's Institute of Hematology and Oncology, Dongguan Taixin Hospital (TXEC-2019-005). This clinical trial was registered at Chinese Clinical Trial Registry (www.chictr.org.cn) with registration number ChiCTR1900027684 and www.clinicaltrials.gov (NCT03222674). Children with R/R AML patients who met criteria for this clinical trial were enrolled. Informed consent according to institutional guidelines and the Declaration of Helsinki was obtained from the parents or guardians. All enrolled patients received one lymphodepleting regimen (cyclophosphamide, 500 - 900 mg/m 2 /day, day -4 to day -1; fludarabine, 25 - 30 mg/m 2 /day, day -2 to day -1 depending on the AML burden) 25 , prior to CAR-T cells infusion. A single dose (0.35-1×10 6 /kg) of anti-CLL1 based CAR-T cells was infused through a peripherally inserted central venous catheter (PICC). The treatment response and CAR-T cell-related toxicities were systemically evaluated. Bone marrow morphologic and flow cytometric assessments for treatment response were performed every month for the first three months after CAR-T cell therapy, and every three months thereafter if not followed by allo-HSCT. Patients achieving CR were transferred for further allo-HSCT if their socioeconomic status and donor status allowed. Generation of clinical-grade CLL1 CAR-T cells All GMP viral vector production practices were following the regulatory guidelines. Lentiviral vector supernatant for the CLL1-CAR was produced by transient transfection of 293T cells (Takeda) with the corresponding CAR plasmid and 3 packaging plasmids: pLP1, pLP2 and pLP/VSVG and the medium was changed 4 hours after transfection. Forty-eight hours later, the cell supernatant was pooled and filtered with a 0.45μm filter, followed by Benzonase treatment (Merck) for 16 hours. Then, the harvest was passed through a Mustang Q ion-exchange capsule (Pall, Ann Arbor, MI). The Mustang Q membrane was washed using 50mM Tris-HCl, pH 8.0 with 750mM NaCl and then eluted in fractions using 50mM Tris-HCL, pH 8.0 with 1.5 M NaCl and diluted with phosphate buffer pH 7.2. The elution was further concentrated approximately 10- fold by 300 KD TFF column. The final concentrate was formulated with human serum albumin (HSA) to 2%, filtered with a 0.22 um filter, aliquoted to 2ml cryotubes, quick frozen on dry-ice, and stored at –80℃. Patients’ T cells from peripheral blood mononuclear cells (PBMCs) were enriched by CD3 magnetic beads (Miltenyi), and stimulated by anti-CD3/CD28 beads (Dynabeads, Human T Activator CD3/CD28, Life Technologies) at a 1:3 (beads: T cells) ratio, and then cultured in H3 medium (Takara) with 4% Human AB serum and 10 ng/mL recombinant human IL7 and IL15 (Miltenyi). Cells were exposed to lentivirus containing supernatant on days 2 and 3 with multiplicity of infection (MOI) of five, on Retronectin-coated non-tissue culture plates (Takara/Clonetech). Beads were magnetically removed on day 4 or 5, and cells were further expanded for 3-5 days in H3 media containing 10 ng/mL recombinant human IL7 and IL15 until use in vitro or in vivo . The cells were harvested and cryo-preserved. Once the standard operating procedure was completed, the cell product was shipped for clinical application under the clinical trial guideline. The ex vivo and in vivo AML targeting capacity of anti-CLL1 based CAR-T cell therapy In vitro cytotoxic assays were performed by co-culturing 50,000 CAR-T cells with 50,000 AML cells in complete media in a 96 well plate. The supernatant was collected after 24h co-incubation. Human interferon gamma (IFNγ) from cell culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA) development kit (4A Biotech, Beijing) according to the manufacturer’s instructions. Severe immune-deficient B-NDG mice were purchased from Jiangsu Biocytogen Co., Ltd. (Nantong, China), and anesthetized with 3% isoflurane (Minrad International, Buffalo, NY, USA) in an incubation chamber. Anesthesia (David Kopf Instruments, Tujunga, CA, USA) was maintained at 2% isoflurane delivered through a nose adaptor. Half million AML HL60-luc cells were injected into 6-10 week-old B-NDG mice using a blunt-end needle through tail vein. Leukemia occurrence was serially monitored by bioluminescence in vivo imaging on an IVIS spectrum instrument (Caliper Life Science) and quantified with Living Image software (PerkinElmer, Waltham, MA, USA). With the establishment of HL60-luc AML B-NDG mice model, mice were randomized and treated with 3 million or 10 million CLL1-CAR-T cells or an equivalent number of non-CAR T cells (matched for total T cell dose) intravenously by tail vein injection. Living Image software was used to analyze the IVIS data. Monitoring cytokine release Novus Biologicals human IL-6 ELISA kit was used to determine the IL6 levels in patients’ plasma. Treatment response evaluation Bone marrow (BM) specimens were longitudinally collected prior to and after CLL1-CAR-T cell infusion for experiments. The National Comprehensive Cancer Network (NCCN) AML Clinical Practice Guidelines Version 3.2021 were used to evaluate the treatment response 26 . Safety and tolerability The common terminology criteria for adverse events (CTCAE) 5.0 criteria was applied to systemically evaluate the safety and tolerability of anti-CLL1 CAR-T cells in this study. All patients were managed using guidelines from Mahadeo and Neelapu 27,28 . Statistical analyses The safety, tolerability, side effects, and clinical response data of patients who received anti-CLL1 CAR-T cells were pooled for analysis using SAS® Version 9.2 or higher, and descriptive statistics were used to summarize the data. Results We initiated a single-arm, Phase 1/2 clinical trial to test the feasibility and safety of the autologous T cells, expressing a CLL1-targeted CAR with 4-1BB and CD3z endoplasmic domains, in patients with refractory/relapsed (R/R) AML. The human CLL1 CAR-T extracellular scFv was derived from a high-affinity CLL1 monoclonal antibody (clone 27H4, Supplementary Fig. 1A ), which was generated by hybridoma technology. Membrane protein array was applied to confirm the CLL1 binding specificity of clone 27H4 as shown in Supplementary Fig. 1B . Then we proceeded to construct the second generation of CAR using the variable region derived from clone 27H4 targeting CCL1 ( Supplementary Fig. 2A ). The design, development and generation of clinical grade anti-CLL1 based CAR-T cells were shown in Supplementary Fig. 2B . Firstly, we tested their anti-tumor efficacy by co-culturing anti-CLL1 CAR-T cells with Raji-CLL1 (B lymphoblastoid cell line ectopically expressing CLL1), or HL60 (CLL1-expressing AML cell line). After co-culturing, a significant IFN-γ release was observed when CLL1-CAR-T cells co-cultured with Raji-CLL1, or HL60 cells, but not with the parental Raji cells which do not express CLL1 ( Supplementary Fig. 3 ), these results demonstrating the targeting capacity of the CLL1-CAR-T cells. Next, we investigated the tumor killing efficacy using human AML-bearing B-NDG mouse model. An increasing dose of anti-CLL1 CAR-T cells (3 × 10 6 and 1 × 10 7 ) were infused into HL60-luc AML-bearing B-NDG mice and the leukemic burden was evaluated by in vivo imaging methods. As shown in Fig. 1 A-B, the AML burden was significantly reduced in CLL1-CAR-T cells-treated group, when compared to their control groups (vehicle and T-mock). Moreover, infusion of CLL1-CAR-T cells, but not the mock T cells, significantly prolonged the survival of the AML-bearing mice, confirming the tumor killing potential of the CLL1-CAR-T cells in vivo (Fig. 1 C). To initiate the Phase 1/2 clinical trial, 8 children with R/R-AML were enrolled; 6 were male; median age was 12 (range 8–16) years (Table 1 ). According to the clinical trial protocol, lymphodepletion conditioning was applied on days − 4, −3, − 2 and − 1 (cyclophosphamide, 500–900 mg/m 2 /day) and days − 2 and − 1 (fludarabine, 25–30 mg/m 2 /day) followed by anti-CLL1 based CAR-T cell infusion on day 0, except patient 2 without lymphodepletion conditioning. The general time schedule of CLL1-CAR-T clinical trial was shown in Fig. 2 A. The therapeutic responses at 1, 2, 3, 6, 9 and 12 months after infusion were assessed by bone marrow morphology and multiple flow cytometry analysis. Detailed patient characteristics and individual CAR-T product information were summarized in Table 1 . The CLL1 expression on AML blasts were determined by flow cytometry using anti-CLL1 antibody. The positivity of CLL1 in AML blasts ranged from 65–96% among these patients (Table 1 ). Patients 1, 3, 5–8 received 1 million CLL1-CAR-T cells per kilogram, patient 2 received 0.35 million CLL1-CAR-T cells per kilogram, and patient 4 received 0.8 million CLL1-CAR-T cells per kilogram (Table 1 ). Table 1 Patient information Patient # Sex/ Age AML subtype Mutation Prior Treatment CLL1% in Tumor Cells CAR-T Product Product CAR% CAR-T Dosage 1 Male/ 16Y AML-M2 RUNX1, FLT-ITD+, NRAS+, U2AF1+ Relapse after HSCT 96% CLL1 43.2% 1×10 6 /kg 2 Male/ 11Y AML-M5 + JMML FLT3+ Relapse after HSCT 95% CLL1 12% 0.35×10 6 /kg 3 Male/ 13Y AML-M2 AML1-ETO fusion, WT1 high expression 5 chemo 86% CLL1 31.3% 1×10 6 /kg 4 Female/ 12Y AML-M2 AML1-ETO fusion, c-KIT mutation 4 chemo 91% CLL1 42.9% 0.8×10 6 /kg 5 Male/ 8Y AML-M6 N/A 2 chemo 95% CLL1 40.8% 1×10 6 /kg 6 Female/ 13Y AML-M2 NUP98DDX10+, KRAS/ETV6/GATA2/W1 /SETD2/MYCN/KDM5C 5 chemo+ Venetoclex 75% CLL1 39.5% 1×10 6 /kg 7 Male/ 12Y AML-M2A PHF6, IDH1, TET2 2 chemo 65% CLL1 67.6% 1×10 6 /kg 8 Male/ 9Y AML-M5 N/A 4 chemo 96% CLL1 60.7% 1×10 6 /kg In “Prior Treatment”, # chemo indicates the number of course of chemotherapy has been taken on the patient. Adverse events (AEs) and clinical outcomes were monitored from the starting point of lymphodepletion until 60 days after CAR-T cell infusion according to the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0. As summarized in Table 2 , all CLL-1 based CAR-T cells treated patients experienced grade 1–2 CRS in the first month. Satisfyingly, no immune effector cell-associated neurotoxicity syndrome (ICANS) was recorded among these patients. To further confirm the CRS/ICANS, we had continuously monitored the pro-inflammatory cytokine IL-6 release. As shown in Supplementary Fig. 4 , the serum IL-6 levels were significantly increased in the first month post CAR-T infusion, which was consistent with the occurrence of CRS. To identify the kinetics of CAR-T cells in patients, we performed flow cytometry analysis to monitor the CAR + T cell expansion. The CAR-T cells were efficiently expanded in the first month after infusion, with the average peak time varied from day 10 to 12, and sustained in vivo for varied time in different individuals ( Supplementary Fig. 5 ). In addition to CRS, we also found that all patients experienced grade 3–4 pancytopenia and absence of monocytes and granulocytes with varied duration (with the shortest for 7 days, and longest for 49 days) following CAR-T infusion. Among these 8 patients, the longest absence of monocytes was 7 weeks in Patient 5. Next, the toxicity profiles of the CAR-T cell infusion were evaluated according to the criteria of CTCAE 5.0. Notably, treatment of CLL1-CAR-T cells had no observable toxicity on all the organs we examined in these 8 patients (Table 2 ). From this point, we concluded that the anti-CLL1 based CAR-T therapy was well tolerated, with the similar toxicity profiles as previously reported using CLL1-CAR-T cells 24 , 25 . Table 2 Patient safety information Patient # Product CRS Myeloablation Cardiac Respiratory Renal Liver Gastrointestine Dermatology 1 CLL1 Grade 1 Yes No No No No No No 2 CLL1 Grade 1 Yes No No No No No No 3 CLL1 Grade 2 Yes No No No No No No 4 CLL1 Grade 2 Yes No No No No No No 5 CLL1 Grade 2 Yes No No No No No No 6 CLL1 Grade 1 Yes No No No No No No 7 CLL1 Grade 1 Yes No No No No No No 8 CLL1 Grade 1 Yes No No No No No No We next assessed the efficacy profiles in these 8 patients with R/R-AML after CLL1-CAR-T treatment. As shown in Fig. 2 B and summarized in Table 3 , morphologic CR (CRm) and MRD negativity were achieved in 4 patients (patient 1, 2, 5, and 8), CRm and MRD positivity were achieved in 2 patients (patient 3 and 7), and partial remission (PR) was achieved in patient 4, and patient 6 remained stable disease (SD) until 1 month after CLL1-CAR-T therapy. To define the long-term effect of anti-CLL1 based CAR-T therapy, we performed a long-term follow-up study among these patients. Among these R/R AML patients, 6 patients (patient 1, 2, 3, 4, 7 and 8) completed allo-HSCT (Fig. 2 B and Table 3 ) after CAR-T treatment. The patient 2 relapsed two months post HSCT and died of GVHD. The patient 4 relapsed six months post HSCT and died of PD. The remaining 4 patients (patient 1, 3, 7 and 8) were still alive and remained complete remission, with the longest 26 months’ follow-up study. The patient 5 remained CR for twelve months without receiving allo-HSCT before relapse. For the patient with stable disease (patient 6) after CAR-T infusion, the percentage of AML blasts decreased from 91.3–70.6% two weeks after CAR-T cell therapy, while the remaining AML blasts were CLL1 negative and CD33 positive ( Supplementary Fig. 6 ). The patient 6 only survived for three months after CAR-T treatment and succumbed to disease progression and lung infection. To summarize the interim analysis of the phase 1/2 clinical trial, the anti-CLL1 CAR-T cells displayed an ideal targeting capacity for treating the pediatric R/R AML patients, and should be considered as an alternative strategy for the treatment of AML in the future. Table 3 Patients’ responses to anti-CLL1-CAR T cells Patient # Sex/ Age AML subtype AML baseline prior to pre-conditioning Product CAR-T Dosage Best response within 1 month Following HSCT Current status 1 Male/ 16Y AML-M2 62.3% CLL1 1×10 6 /kg CR/MRD- Yes CR/MRD- 2 Male/ 11Y AML-M5 + JMML 92.4% CLL1 0.35×10 6 /kg CR/MRD- Yes Death (GVHD) 3 Male/ 13Y AML-M2 21.4% CLL1 1×10 6 /kg CR/MRD+ Yes CR/MRD- 4 Female/ 12Y AML-M2 27.7% CLL1 0.8×10 6 /kg PR Yes Death (PD) 5 Male/ 8Y AML-M6 14.2% CLL1 1×10 6 /kg CR/MRD- No Relapsed 6 Female/ 13Y AML-M2 91.3% CLL1 1×10 6 /kg SD No Death (Lung infection) 7 Male/ 12Y AML-M2A 30% CLL1 1×10 6 /kg CR/MRD+ Yes CR/MRD- 8 Male/ 9Y AML-M5 69.5% CLL1 1×10 6 /kg CR/MRD- Yes CR/MRD- Discussions The application of CD19-directed CAR-T cell therapy has achieved great success for the cure of relapsed or refractory B-cell malignancies (i.e., B-cell acute lymphoblastic leukemia, B-cell lymphoma), also ignited the hope of the treatment of other hematological malignancies. However, the translation of CAR-T cells into AML treatment remains lagged behind, which resulting in immoderate reliance on intensified chemotherapy and allo-HSCT. Here, we reported the safety and efficacy profiles of anti-CLL1-based CAR-T cell therapy in eight children with R/R-AML from two independent medical centers. Up to now, CAR-T cells targeting CLL1, CD13, CD33, TIM3, NKG2D, CD123, CD7, NPM1, and FLT3 have been extensively studied and shown to effectively eradicate AML cells in vitro and in vivo 29 . However, the number of successful case reports using CAR-T cells in treating AML patients is limited 30 . For example, NKG2D-targeted CAR-T has been reported to induce CR in one AML patient 31 . Nevertheless, this successful case cannot be replicated later in a large cohort of AML patients. In a dose-escalation Phase 1 clinical trial, five AML patients treated with high-dose of CD123-CAR-T cells from Mustang Bio Inc, two patients achieved CR and the other three remained SD. It has been reported that CLL1-CD33 compound CAR-T cells treated 9 AML patients, 7 of them reached CR. In addition, two successful case reports have utilized CLL1 as single target of CAR-T therapy, suggesting the promising potential of targeting CLL1 in AML treatment 24 25 . Here, our data further support the efficacy of anti-CLL1-based CAR-T cells in R/R-AML treatment. 7 out of 8 enrolled patients responded to the treatment with only grade1-2 CRS. The CR rate was 75%, PR rate was 12.5% and SD rate was 12.5%. The most severe adverse events associated with this treatment was myeloablation. Meanwhile, no other systemic toxicities were recorded in this study cohort. Together, our findings highlighted the fact that the effective and safe characteristics of anti-CLL1-based CAR-T cell therapy in children with R/R-AML. Several studies have shown that the un-biased tumor associated antigens (TAAs) targeting by CAR-T cells also damaged antigen-expressed healthy tissues (albeit at low levels). For example, treatment with anti-CD123, CD33 CAR-T cells generated a potent anti-AML efficacy while causes severe/prolonged myelosuppression 34 . It has been reported that CLL1 is preferentially highly expressed on AML-stem cells but not on normal HSC, which make it a promising target for AML immunotherapy, although it is also expressed on granulocytes and monocytes 35 . In this study, we did observe severe myelosuppression in all responding cases. Among these patients, patient 5 experienced prolonged myelosuppression, which could not be simply explained by prior lymphodepletion therapy, indicating a higher susceptibility to intracellular bacteria and parasitic infection. For safety, to monitor intracellular bacteria and parasitic infection will be needed for anti-CLL1-based CAR-T therapy. To reduce the risk of infection, we do recommend patients to bridge allo-HSCT once CR was achieved. In addition, we observed that patient 6 poorly responded to anti-CLL1 based CAR-T cells therapy, however, the CLL1 positive AML cells were completely abolished while the remaining AML blasts were CLL1 negative. For the patients with similar outcomes as patient 6, combination of a second CAR-T target depending on the AML immunophenotypic characteristics, such as CD33, CD123, Lewis Y, or CD38, might be helpful in the future to improve the CAR-T cell efficacy. How to balance the risk and benefit when using the CAR-T therapy for R/R-AML treatment remains an important issue in the future. To this end, our results suggested that patients with more than 90% of CLL1 positivity on AML blasts may benefit from single CLL1-CAR-T treatment. However, for patients with less than 90% of CLL1 positivity on AML blasts, an additional CAR to target a different tumor antigen may be beneficial to achieve complete deletion of AML cells. In summary, our findings demonstrate a very encouraging outcome with a safe and manageable profile and high targeting efficacy for the use of anti-CLL1-based CAR-T cells in the treatment of children with R/R-AML. This is the first report of multi-center based clinical trial for application of anti-CLL1 based CAR-T cells in R/R-AML. Declarations Competing Interests statement GL, ZZ, WD, YZ and ML are employees of Guangzhou Bio-Gene Technology Co., Ltd., while other authors have nothing to disclose. Funding This work was partially funded by research funds from St. Baldrick’s Foundation International Scholar (581580), Guangzhou Women and Children’s Medical Center Internal Program (IP-2018-001), and Pearl River S&T Nova Program of Guangzhou (201906010056). Acknowledgements We would like to thank all the patients and their parents for their participation. This work was partially funded by research funds from St. Baldrick’s Foundation International Scholar (581580), Guangzhou Women and Children’s Medical Center Internal Program (IP-2018-001), and Pearl River S&T Nova Program of Guangzhou (201906010056). Author Contributions The study was conceived by HZ and CL, designed by HZ, CL, and ML, supervised by HZ, CL, and ML. HZ, CB, ZP, GL and CL performed the research. HZ, CB, ZP, YH, ZH, and KP recruited the patients and collected clinical data. Data was conducted and interpreted by HZ, CB, ZP, GL, ZZ, WD, ML and CL. HZ, CL, YZ and ML wrote the manuscript. All authors approved the final version for publication. Competing Interests GL, ZZ, WD, YZ and ML are employees of Guangzhou Bio-Gene Technology Co., Ltd., who have potential interest, while other authors have nothing to disclose. References Bonaventure A, Harewood R, Stiller CA, et al. 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Treatment of Acute Myeloid Leukemia with T Cells Expressing Chimeric Antigen Receptors Directed to C-type Lectin-like Molecule 1. Mol Ther. Sep 6 2017;25(9):2202-2213. Wang J, Chen S, Xiao W, et al. CAR-T cells targeting CLL-1 as an approach to treat acute myeloid leukemia. J Hematol Oncol. Jan 10 2018;11(1):7. Zhang H, Wang P, Li Z, He Y, Gan W, Jiang H. Anti-CLL1 Chimeric Antigen Receptor T-Cell Therapy in Children with Relapsed/Refractory Acute Myeloid Leukemia. Clin Cancer Res. Apr 8 2021. Zhang H, Gan WT, Hao WG, Wang PF, Li ZY, Chang LJ. Successful Anti-CLL1 CAR T-Cell Therapy in Secondary Acute Myeloid Leukemia. Frontiers in oncology. 2020;10:685. Tallman MS, Wang ES, Altman JK, et al. Acute Myeloid Leukemia, Version 3.2019, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. Jun 1 2019;17(6):721-749. Mahadeo KM, Khazal SJ, Abdel-Azim H, et al. Management guidelines for paediatric patients receiving chimeric antigen receptor T cell therapy. Nat Rev Clin Oncol. Jan 2019;16(1):45-63. Neelapu SS, Tummala S, Kebriaei P, et al. Chimeric antigen receptor T-cell therapy - assessment and management of toxicities. Nat Rev Clin Oncol. Jan 2018;15(1):47-62. Hofmann S, Schubert ML, Wang L, et al. Chimeric Antigen Receptor (CAR) T Cell Therapy in Acute Myeloid Leukemia (AML). Journal of clinical medicine. Feb 6 2019;8(2). Mardiana S, Gill S. CAR T Cells for Acute Myeloid Leukemia: State of the Art and Future Directions. Frontiers in oncology. 2020;10:697. Sallman DA, Brayer J, Sagatys EM, et al. NKG2D-based chimeric antigen receptor therapy induced remission in a relapsed/refractory acute myeloid leukemia patient. Haematologica. Sep 2018;103(9):e424-e426. Yu B, Liu D. Gemtuzumab ozogamicin and novel antibody-drug conjugates in clinical trials for acute myeloid leukemia. Biomarker research. 2019;7:24. Ataca Atilla P, McKenna MK, Tashiro H, et al. Modulating TNFalpha activity allows transgenic IL15-Expressing CLL-1 CAR T cells to safely eliminate acute myeloid leukemia. Journal for immunotherapy of cancer. Sep 2020;8(2). Petrov JC, Wada M, Pinz KG, et al. Compound CAR T-cells as a double-pronged approach for treating acute myeloid leukemia. Leukemia. Jun 2018;32(6):1317-1326. Ma H, Padmanabhan IS, Parmar S, Gong Y. Targeting CLL-1 for acute myeloid leukemia therapy. J Hematol Oncol. Apr 24 2019;12(1):41. Additional Declarations Yes there is potential conflict of interest. Supplementary Files CLL1CARTSupplementalFigure1Leukemia.pdf CLL1 monoclonal antibody characterization. (A) CLL1 monoclonal antibody affinity analysis, clone 27H4 was selected for further analysis. (B) Membrane protein array using ~6,000 membrane proteins (Integral molecular, PA, USA) identified that clone 27H4 ScFv-human IgG1 Fc specifically bind to CLL1. The other genes above the threshold, including FCGR1A, FCGR3B and FCGR2B, have been shown to have non-specific binding to the IgG1 Fc part. CLL1CARTSupplementalFigure2Leukemia.pdf Generation of anti-CLL1-based CAR-T cells. (A) Schematic of the recombinant lenti-viral vectors of CLL1-CAR. (B) Flowchart of anti-CLL1 CAR-T generation in this study. CLL1CARTSupplementalFigure3Leukemia.pdf Cytotoxicity of CLL1-CAR-T cells. IFN-γ release when CAR-T cells co-cultured with Raji-CLL1, HL60 at an E: T ratio of 1: 1. Time=24 h (n=3). ***P < 0.001. CLL1CARTSupplementalFigure4Leukemia.pdf IL-6 levels in patients’ plasma samples after CAR-T infusion. Blood was drawn from the patients at the indicated time points after CAR-T infusion, plasma was separated and IL-6 levels in the plasma were evaluated by ELISA. CLL1CARTSupplementalFigure5Leukemia.pdf Expansion of CAR-T cells in patients after infusion. Flow cytometry analysis showing the percentage of CAR-T cells in the total T cells in the peripheral blood of AML patients after CAR-T infusion. CLL1CARTSupplementalFigure6Leukemia.pdf CLL1 + AML blasts were cleared by CLL1-CAR-T cells. Flow cytometry analysis showing the AML blast phenotypes changed after CAR-T infusion. The AML blasts transformed to CLL1 negative after anti-CLL1 CAR-T infusion in Patient 6. Cite Share Download PDF Status: Published Journal Publication published 23 Sep, 2022 Read the published version in Leukemia → Version 1 posted Editorial decision: revise 08 Jun, 2022 Review # 2 received at journal 07 Jun, 2022 Reviewer # 2 agreed at journal 23 May, 2022 Review # 1 received at journal 23 May, 2022 Reviewer # 1 agreed at journal 22 May, 2022 Reviewers invited by journal 21 May, 2022 Editor assigned by journal 18 May, 2022 Submission checks completed at journal 18 May, 2022 First submitted to journal 18 May, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1668440","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":107788793,"identity":"23bab1fa-709a-4027-a410-82ceca3240b7","order_by":0,"name":"Min Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYDACdiD+wMCQwEa8FmYGBsYZJGth5gFqIV6HbjOP4WObX3V5fOztFx8XVDDI84sdwK/F7DCPsXFuH1sxG8+ZYuMZZxgMZ84mYCNQi5l0bg9PYptETpo0bxtDgsFtYrRY9kiAtKT/5v1HrBaGHwZALenHmHkbiNLCVmzY25AA8guzNM8xCSL8crx544Mff+ry5NvbH37mqbGR55cmoAUMGNtAJI8BkJAgQjkY/AER7A+IVT4KRsEoGAUjDAAAKOA8V4xOzXkAAAAASUVORK5CYII=","orcid":"","institution":"Guangzhou Bio-gene Technology Co., Ltd","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Luo","suffix":""},{"id":107788794,"identity":"a9f75414-7c29-48db-9976-db6307c4f50a","order_by":1,"name":"HUI ZHANG","email":"","orcid":"","institution":"GUANGZHOU WOMEN AND CHILDREN'S MEDICAL CENTER","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"HUI","middleName":"","lastName":"ZHANG","suffix":""},{"id":107788795,"identity":"3853b12d-3933-405b-8f86-f7d094101e8c","order_by":2,"name":"Chaoke Bu","email":"","orcid":"","institution":"Nanfang-Chunfu Children’s Institute of Hematology and Oncology, Taixin Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chaoke","middleName":"","lastName":"Bu","suffix":""},{"id":107788796,"identity":"58c62721-bf38-4b4d-b4a4-046901834477","order_by":3,"name":"Zhiyong Peng","email":"","orcid":"","institution":"Nanfang-Chunfu Children’s Institute of Hematology and Oncology, Taixin Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhiyong","middleName":"","lastName":"Peng","suffix":""},{"id":107788797,"identity":"152f26c5-0c35-49a6-81b6-2906152a93c4","order_by":4,"name":"Guangchao Li","email":"","orcid":"","institution":"Guangzhou Bio-gene Technology Co., Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guangchao","middleName":"","lastName":"Li","suffix":""},{"id":107788798,"identity":"e621a406-14c5-4345-8008-625a25cb0145","order_by":5,"name":"Zhao Zhou","email":"","orcid":"","institution":"Guangzhou Bio-gene Technology Co., Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhao","middleName":"","lastName":"Zhou","suffix":""},{"id":107788799,"identity":"a0c05e3a-24de-40a5-a465-5dc635ab878c","order_by":6,"name":"Wen Ding","email":"","orcid":"","institution":"Guangzhou Bio-gene Technology Co., Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Ding","suffix":""},{"id":107788800,"identity":"34f0166e-f062-4097-b143-f93bc941b49d","order_by":7,"name":"Yongwei Zheng","email":"","orcid":"","institution":"Guangzhou Bio-gene Technology Co., Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yongwei","middleName":"","lastName":"Zheng","suffix":""},{"id":107788801,"identity":"a46cb7ea-b461-40bf-93e6-6d9167f5f55f","order_by":8,"name":"YINGYI HE","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"YINGYI","middleName":"","lastName":"HE","suffix":""},{"id":107788802,"identity":"16553c04-469b-4ada-8a3d-b157cc184925","order_by":9,"name":"ZHENGBIN HU","email":"","orcid":"","institution":"GUANGZHOU WOMEN AND CHILDREN'S MEDICAL CENTER","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"ZHENGBIN","middleName":"","lastName":"HU","suffix":""},{"id":107788803,"identity":"cedadbf6-6422-4c21-9c83-daab1f8973d6","order_by":10,"name":"Kunlin Pei","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kunlin","middleName":"","lastName":"Pei","suffix":""},{"id":107788804,"identity":"2b694a59-2700-422c-9f1c-6301a6190675","order_by":11,"name":"Chunfu Li","email":"","orcid":"","institution":"Nanfang-Chunfu Children’s Institute of Hematology and Oncology, Taixin Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chunfu","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2022-05-18 08:01:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1668440/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1668440/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41375-022-01703-0","type":"published","date":"2022-09-23T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":21788391,"identity":"40dfb30b-d62d-4d07-97c8-b05e2d777231","added_by":"auto","created_at":"2022-05-23 15:57:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1005561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCLL1-CAR-T showed strong anti-tumor activity in pre-clinical mouse model.\u003c/strong\u003e (A) B-NDG mice were i.v. injected with 5 x10\u003csup\u003e5\u003c/sup\u003e HL60-luciferase cells. After 2 days, the mice were injected intravenously with 3 x10\u003csup\u003e6 \u003c/sup\u003eor 1 x10\u003csup\u003e7\u003c/sup\u003e CLL1-CAR T cells. The BLIs indicated the mouse tumor burden at different time points. (B-C) Tumor burden and survival analysis of mice treated with CLL1-CAR-T cells. (n = 5 mice per group).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"CLL1CARTFigure1Leukemia.png","url":"https://assets-eu.researchsquare.com/files/rs-1668440/v1/1d8d44b435c904636d91896e.png"},{"id":21788389,"identity":"2f9982ff-90bf-4c8f-87f2-d3a9aa7fae3b","added_by":"auto","created_at":"2022-05-23 15:57:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCLL1 CAR-T induced complete remission in AML patients.\u003c/strong\u003e (A) Schematic of the Phase I/II clinical trial design. (B) Duration of the response and overall survival after the infusion of anti-CLL1 based CAR-T cells. 6 out of 8 patients received allo-HSCT after CAR-T cell infusion. Patient 2 died from GVHD after second HSCT. Patient 4 relapsed and died of PD post HSCT. For the other 2 patients who did not receive allo-HSCT, patient 5 relapsed after twelve months remission, patient 6 died from uncontrolled lung infection. CR/CRi: complete response/incomplete response; MRD: minimal residual disease; PR: partial response; SD: stable disease.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"CLL1CARTFigure2Leukemia.png","url":"https://assets-eu.researchsquare.com/files/rs-1668440/v1/daaa310d71211e26d23e57ee.png"},{"id":26912070,"identity":"4f0f7aa0-e069-4ffd-a740-437b7e105617","added_by":"auto","created_at":"2022-09-24 07:06:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":898440,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1668440/v1/f81b3d82-26e4-4724-b55c-4ab8d8f3bb23.pdf"},{"id":21788388,"identity":"f77972ce-1f1e-409f-a2a0-4c7d7370fc0a","added_by":"auto","created_at":"2022-05-23 15:57:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":301758,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCLL1 monoclonal antibody characterization.\u003c/strong\u003e (A) CLL1 monoclonal antibody affinity analysis, clone 27H4 was selected for further analysis. (B) Membrane protein array using ~6,000 membrane proteins (Integral molecular, PA, USA) identified that clone 27H4 ScFv-human IgG1 Fc specifically bind to CLL1. The other genes above the threshold, including FCGR1A, FCGR3B and FCGR2B, have been shown to have non-specific binding to the IgG1 Fc part.\u0026nbsp;\u003c/p\u003e","description":"","filename":"CLL1CARTSupplementalFigure1Leukemia.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1668440/v1/ebb1af951413beb85879519c.pdf"},{"id":21788395,"identity":"1df33dee-da91-416d-ac79-01ccd183a8f6","added_by":"auto","created_at":"2022-05-23 15:57:44","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":181870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of anti-CLL1-based CAR-T cells. \u003c/strong\u003e(A) Schematic of the recombinant lenti-viral vectors of CLL1-CAR. (B) Flowchart of anti-CLL1 CAR-T generation in this study.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"CLL1CARTSupplementalFigure2Leukemia.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1668440/v1/91db977887a37d7b1cb741b4.pdf"},{"id":21788390,"identity":"7b79dc85-6c12-4e77-b03f-cb71e7f03a17","added_by":"auto","created_at":"2022-05-23 15:57:44","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":442143,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxicity of CLL1-CAR-T cells.\u003c/strong\u003e IFN-γ release when CAR-T cells co-cultured with Raji-CLL1, HL60 at an E: T ratio of 1: 1. Time=24 h (n=3). ***P \u0026lt; 0.001.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"CLL1CARTSupplementalFigure3Leukemia.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1668440/v1/11ff953fead342630e930149.pdf"},{"id":21788392,"identity":"6e082a05-8308-499a-9a79-81223d4b8cfa","added_by":"auto","created_at":"2022-05-23 15:57:44","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":184775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-6 levels in patients’ plasma samples after CAR-T infusion\u003c/strong\u003e. Blood was drawn from the patients at the indicated time points after CAR-T infusion, plasma was separated and IL-6 levels in the plasma were evaluated by ELISA.\u003c/p\u003e","description":"","filename":"CLL1CARTSupplementalFigure4Leukemia.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1668440/v1/63d3806a2fdc71812025f177.pdf"},{"id":21788394,"identity":"439a44b8-d1bd-4d8b-a2f2-c62327f0eb5e","added_by":"auto","created_at":"2022-05-23 15:57:44","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":215331,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpansion of CAR-T cells in patients after infusion. \u003c/strong\u003eFlow cytometry analysis showing the percentage of CAR-T cells in the total T cells in the peripheral blood of AML patients after CAR-T infusion.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"CLL1CARTSupplementalFigure5Leukemia.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1668440/v1/c4fb3e686dfa94e42272f550.pdf"},{"id":21788844,"identity":"7d1c7b6e-22e3-437d-818d-22e7e85068d8","added_by":"auto","created_at":"2022-05-23 16:02:44","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":428391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCLL1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e AML blasts were cleared by CLL1-CAR-T cells. \u003c/strong\u003eFlow cytometry analysis showing the AML blast phenotypes changed after CAR-T infusion. The AML blasts transformed to CLL1 negative after anti-CLL1 CAR-T infusion in Patient 6.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"CLL1CARTSupplementalFigure6Leukemia.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1668440/v1/d6aea2bc1ceaa2334caa01da.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential conflict of interest.","formattedTitle":"Characteristics of anti-CLL1 based CAR-T therapy for children with relapsed or refractory acute myeloid leukemia: the multi-center efficacy and safety interim analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOutcomes of children with acute myeloid leukemia (AML) remain lagged behind that of children with acute lymphoblastic leukemia (ALL), with 5-year disease-free survival (DFS) varying between 33.3% and 79.5% worldwide, as reported by the CONCORD-2 study\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Though 50% \u0026ndash; 70% of children with primary AML can be cured with conventional intensive therapy\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, the cure rate is not improved much by the introduction of novel agents\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The relative poor prognosis for childhood AML has been mostly attributed to less available targeted agents or therapies for those relapsed/refractory AML (R/R-AML) patients. Up to now, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is generally considered as the best chance of cure for patients with high-risk- or R/R- AML\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImpressively, the prognosis for patients with R/R-AML can be greatly enhanced if they are in a negative minimal residual disease (MRD) state even without receiving allo-HSCT\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Therefore, novel therapies are highly needed for further improvement in the R/R-AML therapy. Several novel agents have been currently introduced into pediatric R/R-AML therapies (i.e., sorafenib, gilterinib, gemtuzumab ozogamicin (GO), and venetoclax), however the responses remain unsatisfied, with ~\u0026thinsp;50% CR/CRi (complete remission/complete remission with incomplete hematologic recovery) rate\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The development of immunotherapy has given the clinicians with new strategy for treating patients with hematological malignancies. In this regard, autologous chimeric antigen receptor (CAR) T cell therapy has been increasingly accepted as a highly effective regimen for relapsed/refractory hematological malignancies\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. For example, anti-CD19 CAR-T cell immunotherapies are reported to be highly effective in R/R B-cell ALL (B-ALL) patients, with CR rates ranging from 70 to 94%\u003csup\u003e14\u0026ndash;16\u003c/sup\u003e, although near one third responding patients eventually experienced relapse due to multiple mechanisms\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, the potential of CAR-T cell therapy in R/R-AML remains undetermined.\u003c/p\u003e \u003cp\u003eC-type lectin‐like molecule‐1 (CLL1) is identified as a novel AML stem cell associated antigen\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Intriguingly, CLL1 is highly expressed on AML leukemia stem cells (LSCs), majority of AML blasts and normal myeloid cells, but not on normal hematopoietic stem cells (HSCs) and lymphoid cells, suggesting that targeting CLL1 can be a novel AML therapy while not affecting normal hematopoiesis and lymphocyte-directed immune function. Several studies have successfully developed novel CLL1-directed therapies (i.e., antibody-based and cellular therapies) with definite efficacy on human AML with promising \u003cem\u003eex vivo\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e evidences\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Liu and Zhang\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e have independently and successfully treated two secondary AML patients with anti-CLL1 based CAR-T cells, highlighting the potential of CAR-T cell therapy in R/R-AML.\u003c/p\u003e \u003cp\u003eTo this end, we recruited 8 children with R/R-AML for a phase 1/2 clinical trial with autologous anti-CLL1 based CAR-T cell therapy to test its safety and efficacy among two independent institutions.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eEligibility, ethics approval, and treatment schema\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board of Guangzhou Women and Children\u0026rsquo;s Medical Center (2020-23) and Nanfang-Chunfu Children's Institute of Hematology and Oncology, Dongguan Taixin Hospital (TXEC-2019-005). This clinical trial was registered at Chinese Clinical Trial Registry (www.chictr.org.cn) with registration number ChiCTR1900027684 and www.clinicaltrials.gov (NCT03222674). Children with R/R AML patients who met criteria for this clinical trial were enrolled. Informed consent according to institutional guidelines and the Declaration of Helsinki was obtained from the parents or guardians. All enrolled patients received one lymphodepleting regimen (cyclophosphamide, 500 - 900 mg/m\u003csup\u003e2\u003c/sup\u003e/day, day -4 to day -1; fludarabine, 25 - 30 mg/m\u003csup\u003e2\u003c/sup\u003e/day, day -2 to day -1 depending on the AML burden)\u003csup\u003e25\u003c/sup\u003e, prior to CAR-T cells infusion. A single dose (0.35-1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg) of anti-CLL1 based CAR-T cells was infused through a peripherally inserted central venous catheter (PICC). The treatment response and CAR-T cell-related toxicities were systemically evaluated. Bone marrow morphologic and flow cytometric assessments for treatment response were performed every month for the first three months after CAR-T cell therapy, and every three months thereafter if not followed by allo-HSCT. Patients achieving CR were transferred for further allo-HSCT if their socioeconomic status and donor status allowed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of clinical-grade CLL1 CAR-T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll GMP viral vector production practices were following the regulatory guidelines. Lentiviral vector supernatant for the CLL1-CAR was produced by transient transfection of 293T cells (Takeda) with the corresponding CAR plasmid and 3 packaging plasmids: pLP1, pLP2 and pLP/VSVG and the medium was changed 4 hours after transfection. Forty-eight hours later, the cell supernatant was pooled and filtered with a 0.45\u0026mu;m filter, followed by Benzonase treatment (Merck) for 16 hours. Then, the harvest was passed through a Mustang Q ion-exchange capsule (Pall, Ann Arbor, MI). The Mustang Q membrane was washed using 50mM Tris-HCl, pH 8.0 with 750mM NaCl and then eluted in fractions using 50mM Tris-HCL, pH 8.0 with 1.5 M NaCl and diluted with phosphate buffer pH 7.2. The elution was further concentrated approximately 10- fold by 300 KD TFF column. The final concentrate was formulated with human serum albumin (HSA) to 2%, filtered with a 0.22 um filter, aliquoted to 2ml cryotubes, quick frozen on dry-ice, and stored at \u0026ndash;80℃.\u003c/p\u003e\n\u003cp\u003ePatients\u0026rsquo; T cells from peripheral blood mononuclear cells (PBMCs) were enriched by CD3 magnetic beads (Miltenyi), and stimulated by anti-CD3/CD28 beads (Dynabeads, Human T Activator CD3/CD28, Life Technologies) at a 1:3 (beads: T cells) ratio, and then cultured in H3 medium (Takara) with 4% Human AB serum and 10 ng/mL recombinant human IL7 and IL15 (Miltenyi). Cells were exposed to lentivirus containing supernatant on days 2 and 3 with multiplicity of infection (MOI) of five, on Retronectin-coated non-tissue culture plates (Takara/Clonetech). Beads were magnetically removed on day 4 or 5, and cells were further expanded for 3-5 days in H3 media containing 10 ng/mL recombinant human IL7 and IL15 until use \u003cem\u003ein vitro\u003c/em\u003e or \u003cem\u003ein vivo\u003c/em\u003e. The cells were harvested and cryo-preserved. Once the standard operating procedure was completed, the cell product was shipped for clinical application under the clinical trial guideline.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe \u003cem\u003eex vivo\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e AML targeting capacity of anti-CLL1 based CAR-T cell therapy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e cytotoxic assays were performed by co-culturing 50,000 CAR-T cells with 50,000 AML cells in complete media in a 96 well plate. The supernatant was collected after 24h co-incubation. Human interferon gamma (IFN\u0026gamma;) from cell culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA) development kit (4A Biotech, Beijing) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003eSevere immune-deficient B-NDG mice were purchased from Jiangsu Biocytogen Co., Ltd. (Nantong, China), and anesthetized with 3% isoflurane (Minrad International, Buffalo, NY, USA) in an incubation chamber. Anesthesia (David Kopf Instruments, Tujunga, CA, USA) was maintained at 2% isoflurane delivered through a nose adaptor. Half million AML HL60-luc cells were injected into 6-10 week-old B-NDG mice using a blunt-end needle through tail vein. Leukemia occurrence was serially monitored by bioluminescence \u003cem\u003ein vivo \u003c/em\u003eimaging on an IVIS spectrum instrument (Caliper Life Science) and quantified with Living Image software (PerkinElmer, Waltham, MA, USA). With the establishment of HL60-luc AML B-NDG mice model, mice were randomized and treated with 3 million or 10 million CLL1-CAR-T cells or an equivalent number of non-CAR T cells (matched for total T cell dose) intravenously by tail vein injection. Living Image software was used to analyze the IVIS data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMonitoring cytokine release\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNovus Biologicals human IL-6 ELISA kit was used to determine the IL6 levels in patients\u0026rsquo; plasma.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment response evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBone marrow (BM) specimens were longitudinally collected prior to and after CLL1-CAR-T cell infusion for experiments. The National Comprehensive Cancer Network (NCCN) AML Clinical Practice Guidelines Version 3.2021 were used to evaluate the treatment response \u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSafety and tolerability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe common terminology criteria for adverse events (CTCAE) 5.0 criteria was applied to systemically evaluate the safety and tolerability of anti-CLL1 CAR-T cells in this study. All patients were managed using guidelines from Mahadeo and Neelapu\u003csup\u003e27,28\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe safety, tolerability, side effects, and clinical response data of patients who received anti-CLL1 CAR-T cells were pooled for analysis using SAS\u0026reg; Version 9.2 or higher, and descriptive statistics were used to summarize the data.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe initiated a single-arm, Phase 1/2 clinical trial to test the feasibility and safety of the autologous T cells, expressing a CLL1-targeted CAR with 4-1BB and CD3z endoplasmic domains, in patients with refractory/relapsed (R/R) AML. The human CLL1 CAR-T extracellular scFv was derived from a high-affinity CLL1 monoclonal antibody (clone 27H4, \u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e), which was generated by hybridoma technology. Membrane protein array was applied to confirm the CLL1 binding specificity of clone 27H4 as shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e. Then we proceeded to construct the second generation of CAR using the variable region derived from clone 27H4 targeting CCL1 (\u003cb\u003eSupplementary Fig.\u0026nbsp;2A\u003c/b\u003e). The design, development and generation of clinical grade anti-CLL1 based CAR-T cells were shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;2B\u003c/b\u003e. Firstly, we tested their anti-tumor efficacy by co-culturing anti-CLL1 CAR-T cells with Raji-CLL1 (B lymphoblastoid cell line ectopically expressing CLL1), or HL60 (CLL1-expressing AML cell line). After co-culturing, a significant IFN-γ release was observed when CLL1-CAR-T cells co-cultured with Raji-CLL1, or HL60 cells, but not with the parental Raji cells which do not express CLL1 (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e), these results demonstrating the targeting capacity of the CLL1-CAR-T cells. Next, we investigated the tumor killing efficacy using human AML-bearing B-NDG mouse model. An increasing dose of anti-CLL1 CAR-T cells (3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e and 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e) were infused into HL60-luc AML-bearing B-NDG mice and the leukemic burden was evaluated by \u003cem\u003ein vivo\u003c/em\u003e imaging methods. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B, the AML burden was significantly reduced in CLL1-CAR-T cells-treated group, when compared to their control groups (vehicle and T-mock). Moreover, infusion of CLL1-CAR-T cells, but not the mock T cells, significantly prolonged the survival of the AML-bearing mice, confirming the tumor killing potential of the CLL1-CAR-T cells \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo initiate the Phase 1/2 clinical trial, 8 children with R/R-AML were enrolled; 6 were male; median age was 12 (range 8\u0026ndash;16) years (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). According to the clinical trial protocol, lymphodepletion conditioning was applied on days \u0026minus;\u0026thinsp;4, \u0026minus;3, \u0026minus;\u0026thinsp;2 and \u0026minus;\u0026thinsp;1 (cyclophosphamide, 500\u0026ndash;900 mg/m\u003csup\u003e2\u003c/sup\u003e/day) and days \u0026minus;\u0026thinsp;2 and \u0026minus;\u0026thinsp;1 (fludarabine, 25\u0026ndash;30 mg/m\u003csup\u003e2\u003c/sup\u003e/day) followed by anti-CLL1 based CAR-T cell infusion on day 0, except patient 2 without lymphodepletion conditioning. The general time schedule of CLL1-CAR-T clinical trial was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. The therapeutic responses at 1, 2, 3, 6, 9 and 12 months after infusion were assessed by bone marrow morphology and multiple flow cytometry analysis. Detailed patient characteristics and individual CAR-T product information were summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The CLL1 expression on AML blasts were determined by flow cytometry using anti-CLL1 antibody. The positivity of CLL1 in AML blasts ranged from 65\u0026ndash;96% among these patients (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Patients 1, 3, 5\u0026ndash;8 received 1\u0026nbsp;million CLL1-CAR-T cells per kilogram, patient 2 received 0.35\u0026nbsp;million CLL1-CAR-T cells per kilogram, and patient 4 received 0.8\u0026nbsp;million CLL1-CAR-T cells per kilogram (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient information\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient #\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex/\u003c/p\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML subtype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMutation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePrior\u003c/p\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCLL1% in Tumor Cells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCAR-T Product\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eProduct CAR%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCAR-T Dosage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale/\u003c/p\u003e \u003cp\u003e16Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRUNX1, FLT-ITD+,\u003c/p\u003e \u003cp\u003eNRAS+, U2AF1+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRelapse after HSCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e96%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e43.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale/\u003c/p\u003e \u003cp\u003e11Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M5\u0026thinsp;+\u0026thinsp;JMML\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFLT3+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRelapse after HSCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.35\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale/\u003c/p\u003e \u003cp\u003e13Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAML1-ETO fusion,\u003c/p\u003e \u003cp\u003eWT1 high expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 chemo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e86%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e31.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale/\u003c/p\u003e \u003cp\u003e12Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAML1-ETO fusion,\u003c/p\u003e \u003cp\u003ec-KIT mutation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 chemo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e91%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e42.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.8\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale/\u003c/p\u003e \u003cp\u003e8Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 chemo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e40.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale/\u003c/p\u003e \u003cp\u003e13Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNUP98DDX10+, KRAS/ETV6/GATA2/W1\u003c/p\u003e \u003cp\u003e/SETD2/MYCN/KDM5C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 chemo+\u003c/p\u003e \u003cp\u003eVenetoclex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e39.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale/\u003c/p\u003e \u003cp\u003e12Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M2A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePHF6, IDH1, TET2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 chemo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e67.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale/\u003c/p\u003e \u003cp\u003e9Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 chemo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e96%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e60.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eIn \u0026ldquo;Prior Treatment\u0026rdquo;, # chemo indicates the number of course of chemotherapy has been taken on the patient.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdverse events (AEs) and clinical outcomes were monitored from the starting point of lymphodepletion until 60 days after CAR-T cell infusion according to the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0. As summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, all CLL-1 based CAR-T cells treated patients experienced grade 1\u0026ndash;2 CRS in the first month. Satisfyingly, no immune effector cell-associated neurotoxicity syndrome (ICANS) was recorded among these patients. To further confirm the CRS/ICANS, we had continuously monitored the pro-inflammatory cytokine IL-6 release. As shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e, the serum IL-6 levels were significantly increased in the first month post CAR-T infusion, which was consistent with the occurrence of CRS. To identify the kinetics of CAR-T cells in patients, we performed flow cytometry analysis to monitor the CAR\u003csup\u003e+\u003c/sup\u003e T cell expansion. The CAR-T cells were efficiently expanded in the first month after infusion, with the average peak time varied from day 10 to 12, and sustained \u003cem\u003ein vivo\u003c/em\u003e for varied time in different individuals (\u003cb\u003eSupplementary Fig.\u0026nbsp;5\u003c/b\u003e). In addition to CRS, we also found that all patients experienced grade 3\u0026ndash;4 pancytopenia and absence of monocytes and granulocytes with varied duration (with the shortest for 7 days, and longest for 49 days) following CAR-T infusion. Among these 8 patients, the longest absence of monocytes was 7 weeks in Patient 5. Next, the toxicity profiles of the CAR-T cell infusion were evaluated according to the criteria of CTCAE 5.0. Notably, treatment of CLL1-CAR-T cells had no observable toxicity on all the organs we examined in these 8 patients (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). From this point, we concluded that the anti-CLL1 based CAR-T therapy was well tolerated, with the similar toxicity profiles as previously reported using CLL1-CAR-T cells\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient safety information\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient #\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProduct\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCRS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMyeloablation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCardiac\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRespiratory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRenal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLiver\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eGastrointestine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eDermatology\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrade 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrade 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrade 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrade 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrade 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrade 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrade 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrade 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe next assessed the efficacy profiles in these 8 patients with R/R-AML after CLL1-CAR-T treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, morphologic CR (CRm) and MRD negativity were achieved in 4 patients (patient 1, 2, 5, and 8), CRm and MRD positivity were achieved in 2 patients (patient 3 and 7), and partial remission (PR) was achieved in patient 4, and patient 6 remained stable disease (SD) until 1 month after CLL1-CAR-T therapy. To define the long-term effect of anti-CLL1 based CAR-T therapy, we performed a long-term follow-up study among these patients. Among these R/R AML patients, 6 patients (patient 1, 2, 3, 4, 7 and 8) completed allo-HSCT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) after CAR-T treatment. The patient 2 relapsed two months post HSCT and died of GVHD. The patient 4 relapsed six months post HSCT and died of PD. The remaining 4 patients (patient 1, 3, 7 and 8) were still alive and remained complete remission, with the longest 26 months\u0026rsquo; follow-up study. The patient 5 remained CR for twelve months without receiving allo-HSCT before relapse. For the patient with stable disease (patient 6) after CAR-T infusion, the percentage of AML blasts decreased from 91.3\u0026ndash;70.6% two weeks after CAR-T cell therapy, while the remaining AML blasts were CLL1 negative and CD33 positive (\u003cb\u003eSupplementary Fig.\u0026nbsp;6\u003c/b\u003e). The patient 6 only survived for three months after CAR-T treatment and succumbed to disease progression and lung infection. To summarize the interim analysis of the phase 1/2 clinical trial, the anti-CLL1 CAR-T cells displayed an ideal targeting capacity for treating the pediatric R/R AML patients, and should be considered as an alternative strategy for the treatment of AML in the future.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatients\u0026rsquo; responses to anti-CLL1-CAR T cells\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient #\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex/\u003c/p\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML subtype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAML baseline prior to pre-conditioning\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProduct\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCAR-T Dosage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBest response within 1 month\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFollowing HSCT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCurrent status\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale/\u003c/p\u003e \u003cp\u003e16Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCR/MRD-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCR/MRD-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale/\u003c/p\u003e \u003cp\u003e11Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M5\u0026thinsp;+\u0026thinsp;JMML\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.35\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCR/MRD-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDeath\u003c/p\u003e \u003cp\u003e(GVHD)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale/\u003c/p\u003e \u003cp\u003e13Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCR/MRD+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCR/MRD-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale/\u003c/p\u003e \u003cp\u003e12Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.8\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDeath\u003c/p\u003e \u003cp\u003e(PD)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale/\u003c/p\u003e \u003cp\u003e8Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCR/MRD-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eRelapsed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale/\u003c/p\u003e \u003cp\u003e13Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDeath\u003c/p\u003e \u003cp\u003e(Lung infection)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale/\u003c/p\u003e \u003cp\u003e12Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M2A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCR/MRD+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCR/MRD-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale/\u003c/p\u003e \u003cp\u003e9Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAML-M5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e69.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCR/MRD-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCR/MRD-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussions","content":"\u003cp\u003eThe application of CD19-directed CAR-T cell therapy has achieved great success for the cure of relapsed or refractory B-cell malignancies (i.e., B-cell acute lymphoblastic leukemia, B-cell lymphoma), also ignited the hope of the treatment of other hematological malignancies. However, the translation of CAR-T cells into AML treatment remains lagged behind, which resulting in immoderate reliance on intensified chemotherapy and allo-HSCT. Here, we reported the safety and efficacy profiles of anti-CLL1-based CAR-T cell therapy in eight children with R/R-AML from two independent medical centers.\u003c/p\u003e \u003cp\u003eUp to now, CAR-T cells targeting CLL1, CD13, CD33, TIM3, NKG2D, CD123, CD7, NPM1, and FLT3 have been extensively studied and shown to effectively eradicate AML cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, the number of successful case reports using CAR-T cells in treating AML patients is limited\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. For example, NKG2D-targeted CAR-T has been reported to induce CR in one AML patient\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Nevertheless, this successful case cannot be replicated later in a large cohort of AML patients. In a dose-escalation Phase 1 clinical trial, five AML patients treated with high-dose of CD123-CAR-T cells from Mustang Bio Inc, two patients achieved CR and the other three remained SD. It has been reported that CLL1-CD33 compound CAR-T cells treated 9 AML patients, 7 of them reached CR. In addition, two successful case reports have utilized CLL1 as single target of CAR-T therapy, suggesting the promising potential of targeting CLL1 in AML treatment\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e 25\u003c/sup\u003e. Here, our data further support the efficacy of anti-CLL1-based CAR-T cells in R/R-AML treatment. 7 out of 8 enrolled patients responded to the treatment with only grade1-2 CRS. The CR rate was 75%, PR rate was 12.5% and SD rate was 12.5%. The most severe adverse events associated with this treatment was myeloablation. Meanwhile, no other systemic toxicities were recorded in this study cohort. Together, our findings highlighted the fact that the effective and safe characteristics of anti-CLL1-based CAR-T cell therapy in children with R/R-AML.\u003c/p\u003e \u003cp\u003eSeveral studies have shown that the un-biased tumor associated antigens (TAAs) targeting by CAR-T cells also damaged antigen-expressed healthy tissues (albeit at low levels). For example, treatment with anti-CD123, CD33 CAR-T cells generated a potent anti-AML efficacy while causes severe/prolonged myelosuppression\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. It has been reported that CLL1 is preferentially highly expressed on AML-stem cells but not on normal HSC, which make it a promising target for AML immunotherapy, although it is also expressed on granulocytes and monocytes\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In this study, we did observe severe myelosuppression in all responding cases. Among these patients, patient 5 experienced prolonged myelosuppression, which could not be simply explained by prior lymphodepletion therapy, indicating a higher susceptibility to intracellular bacteria and parasitic infection. For safety, to monitor intracellular bacteria and parasitic infection will be needed for anti-CLL1-based CAR-T therapy. To reduce the risk of infection, we do recommend patients to bridge allo-HSCT once CR was achieved. In addition, we observed that patient 6 poorly responded to anti-CLL1 based CAR-T cells therapy, however, the CLL1 positive AML cells were completely abolished while the remaining AML blasts were CLL1 negative. For the patients with similar outcomes as patient 6, combination of a second CAR-T target depending on the AML immunophenotypic characteristics, such as CD33, CD123, Lewis Y, or CD38, might be helpful in the future to improve the CAR-T cell efficacy. How to balance the risk and benefit when using the CAR-T therapy for R/R-AML treatment remains an important issue in the future. To this end, our results suggested that patients with more than 90% of CLL1 positivity on AML blasts may benefit from single CLL1-CAR-T treatment. However, for patients with less than 90% of CLL1 positivity on AML blasts, an additional CAR to target a different tumor antigen may be beneficial to achieve complete deletion of AML cells.\u003c/p\u003e \u003cp\u003eIn summary, our findings demonstrate a very encouraging outcome with a safe and manageable profile and high targeting efficacy for the use of anti-CLL1-based CAR-T cells in the treatment of children with R/R-AML. This is the first report of multi-center based clinical trial for application of anti-CLL1 based CAR-T cells in R/R-AML.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGL, ZZ, WD, YZ and ML are employees of\u0026nbsp;Guangzhou Bio-Gene Technology Co., Ltd., while other\u0026nbsp;authors have nothing to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially funded by research funds from St. Baldrick\u0026rsquo;s Foundation International Scholar (581580), Guangzhou Women and Children\u0026rsquo;s Medical Center Internal Program (IP-2018-001), and Pearl River S\u0026amp;T Nova Program of Guangzhou (201906010056).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all the patients and their parents for their participation. This work was partially funded by research funds from St. Baldrick\u0026rsquo;s Foundation International Scholar (581580), Guangzhou Women and Children\u0026rsquo;s Medical Center Internal Program (IP-2018-001), and Pearl River S\u0026amp;T Nova Program of Guangzhou (201906010056).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conceived by HZ and CL, designed by HZ, CL, and ML, supervised by HZ, CL, and ML. HZ, CB, ZP, GL and CL performed the research. HZ, CB, ZP, YH, ZH, and KP recruited the patients and collected clinical data. \u0026nbsp;Data was conducted and interpreted by HZ, CB, ZP, GL, ZZ, WD, ML and CL. HZ, CL, YZ and ML wrote the manuscript. All authors approved the final version for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGL, ZZ, WD, YZ and ML are employees of Guangzhou Bio-Gene Technology Co., Ltd., who have potential interest, while other authors have nothing to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBonaventure A, Harewood R, Stiller CA, et al. Worldwide comparison of survival from childhood leukaemia for 1995-2009, by subtype, age, and sex (CONCORD-2): a population-based study of individual data for 89 828 children from 198 registries in 53 countries. \u003cem\u003eThe Lancet. Haematology. \u003c/em\u003eMay 2017;4(5):e202-e217.\u003c/li\u003e\n\u003cli\u003eRubnitz JE, Inaba H, Dahl G, et al. 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The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells. \u003cem\u003eBlood. \u003c/em\u003eOct 1 2007;110(7):2659-2666.\u003c/li\u003e\n\u003cli\u003eLeong SR, Sukumaran S, Hristopoulos M, et al. An anti-CD3/anti-CLL-1 bispecific antibody for the treatment of acute myeloid leukemia. \u003cem\u003eBlood. \u003c/em\u003eFeb 2 2017;129(5):609-618.\u003c/li\u003e\n\u003cli\u003eJiang YP, Liu BY, Zheng Q, et al. CLT030, a leukemic stem cell-targeting CLL1 antibody-drug conjugate for treatment of acute myeloid leukemia. \u003cem\u003eBlood Adv. \u003c/em\u003eJul 24 2018;2(14):1738-1749.\u003c/li\u003e\n\u003cli\u003eLin TY, Zhu Y, Li Y, et al. Daunorubicin-containing CLL1-targeting nanomicelles have anti-leukemia stem cell activity in acute myeloid leukemia. \u003cem\u003eNanomedicine : nanotechnology, biology, and medicine. \u003c/em\u003eAug 2019;20:102004.\u003c/li\u003e\n\u003cli\u003eTashiro H, Sauer T, Shum T, et al. Treatment of Acute Myeloid Leukemia with T Cells Expressing Chimeric Antigen Receptors Directed to C-type Lectin-like Molecule 1. \u003cem\u003eMol Ther. \u003c/em\u003eSep 6 2017;25(9):2202-2213.\u003c/li\u003e\n\u003cli\u003eWang J, Chen S, Xiao W, et al. CAR-T cells targeting CLL-1 as an approach to treat acute myeloid leukemia. \u003cem\u003eJ Hematol Oncol. \u003c/em\u003eJan 10 2018;11(1):7.\u003c/li\u003e\n\u003cli\u003eZhang H, Wang P, Li Z, He Y, Gan W, Jiang H. Anti-CLL1 Chimeric Antigen Receptor T-Cell Therapy in Children with Relapsed/Refractory Acute Myeloid Leukemia. \u003cem\u003eClin Cancer Res. \u003c/em\u003eApr 8 2021.\u003c/li\u003e\n\u003cli\u003eZhang H, Gan WT, Hao WG, Wang PF, Li ZY, Chang LJ. Successful Anti-CLL1 CAR T-Cell Therapy in Secondary Acute Myeloid Leukemia. \u003cem\u003eFrontiers in oncology. \u003c/em\u003e2020;10:685.\u003c/li\u003e\n\u003cli\u003eTallman MS, Wang ES, Altman JK, et al. Acute Myeloid Leukemia, Version 3.2019, NCCN Clinical Practice Guidelines in Oncology. \u003cem\u003eJ Natl Compr Canc Netw. \u003c/em\u003eJun 1 2019;17(6):721-749.\u003c/li\u003e\n\u003cli\u003eMahadeo KM, Khazal SJ, Abdel-Azim H, et al. Management guidelines for paediatric patients receiving chimeric antigen receptor T cell therapy. \u003cem\u003eNat Rev Clin Oncol. \u003c/em\u003eJan 2019;16(1):45-63.\u003c/li\u003e\n\u003cli\u003eNeelapu SS, Tummala S, Kebriaei P, et al. Chimeric antigen receptor T-cell therapy - assessment and management of toxicities. \u003cem\u003eNat Rev Clin Oncol. \u003c/em\u003eJan 2018;15(1):47-62.\u003c/li\u003e\n\u003cli\u003eHofmann S, Schubert ML, Wang L, et al. Chimeric Antigen Receptor (CAR) T Cell Therapy in Acute Myeloid Leukemia (AML). \u003cem\u003eJournal of clinical medicine. \u003c/em\u003eFeb 6 2019;8(2).\u003c/li\u003e\n\u003cli\u003eMardiana S, Gill S. CAR T Cells for Acute Myeloid Leukemia: State of the Art and Future Directions. \u003cem\u003eFrontiers in oncology. \u003c/em\u003e2020;10:697.\u003c/li\u003e\n\u003cli\u003eSallman DA, Brayer J, Sagatys EM, et al. NKG2D-based chimeric antigen receptor therapy induced remission in a relapsed/refractory acute myeloid leukemia patient. \u003cem\u003eHaematologica. \u003c/em\u003eSep 2018;103(9):e424-e426.\u003c/li\u003e\n\u003cli\u003eYu B, Liu D. Gemtuzumab ozogamicin and novel antibody-drug conjugates in clinical trials for acute myeloid leukemia. \u003cem\u003eBiomarker research. \u003c/em\u003e2019;7:24.\u003c/li\u003e\n\u003cli\u003eAtaca Atilla P, McKenna MK, Tashiro H, et al. Modulating TNFalpha activity allows transgenic IL15-Expressing CLL-1 CAR T cells to safely eliminate acute myeloid leukemia. \u003cem\u003eJournal for immunotherapy of cancer. \u003c/em\u003eSep 2020;8(2).\u003c/li\u003e\n\u003cli\u003ePetrov JC, Wada M, Pinz KG, et al. Compound CAR T-cells as a double-pronged approach for treating acute myeloid leukemia. \u003cem\u003eLeukemia. \u003c/em\u003eJun 2018;32(6):1317-1326.\u003c/li\u003e\n\u003cli\u003eMa H, Padmanabhan IS, Parmar S, Gong Y. Targeting CLL-1 for acute myeloid leukemia therapy. \u003cem\u003eJ Hematol Oncol. \u003c/em\u003eApr 24 2019;12(1):41.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"relapsed, refractory, childhood acute myeloid leukemia, CAR-T cells, CLL1","lastPublishedDoi":"10.21203/rs.3.rs-1668440/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1668440/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eC-type lectin like molecule-1 (CLL1) is preferentially expressed on acute myeloid leukemia (AML) stem cells and AML blasts, and can be considered as AML-associated antigen. Anti-CLL1-based CAR-T cells exhibited effective tumor killing capacity \u003cem\u003ein vitro\u003c/em\u003e and in AML-bearing mouse model. In this report, eight children with relapsed or refractory AML (R/R-AML) were recruited for a phase 1/2 clinical trial of autologous anti-CLL1 CAR-T cell immunotherapy. The objectives of this clinical trial were to evaluate the safety and the anti-AML responses after CLL1-CAR-T cell treatment, with long-term prognosis within those patients who did not receive allogeneic hematopoietic stem cells transplantation (allo-HSCT) as an additional aim. These R/R-AML patients received one dose of autologous CLL1-CAR-T cells after lymphodepletion conditioning. Grade 3\u0026ndash;4 hematologic adverse events were observed post CAR-T cell infusion. Meanwhile, grade 1\u0026ndash;2 cytokine release syndrome (CRS) was observed but without any lethal events. 4 out of 8 AML patients achieved incomplete remission (CRi) and minimal residual disease (MRD) negativity, 2 patients with CRi but MRD positivity, and 2 patients with decreased AML burden and CLL1 positive AML blast clearance. These results suggested that anti-CLL1-based CAR-T cell immunotherapy can be considered as a well-tolerated and effective option for treating children with R/R-AML.\u003c/p\u003e","manuscriptTitle":"Characteristics of anti-CLL1 based CAR-T therapy for children with relapsed or refractory acute myeloid leukemia: the multi-center efficacy and safety interim analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-23 15:57:42","doi":"10.21203/rs.3.rs-1668440/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2022-06-08T09:47:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-06-07T13:46:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-05-23T21:34:27+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-05-23T08:52:00+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-05-22T20:46:27+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2022-05-21T23:39:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-05-18T11:56:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-05-18T11:56:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Leukemia","date":"2022-05-18T07:59:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0e71c9d3-7d27-4023-8ec5-e92e87760291","owner":[],"postedDate":"May 23rd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2022-09-24T07:06:24+00:00","versionOfRecord":{"articleIdentity":"rs-1668440","link":"https://doi.org/10.1038/s41375-022-01703-0","journal":{"identity":"leukemia","isVorOnly":false,"title":"Leukemia"},"publishedOn":"2022-09-23 04:00:00","publishedOnDateReadable":"September 23rd, 2022"},"versionCreatedAt":"2022-05-23 15:57:42","video":"","vorDoi":"10.1038/s41375-022-01703-0","vorDoiUrl":"https://doi.org/10.1038/s41375-022-01703-0","workflowStages":[]},"version":"v1","identity":"rs-1668440","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1668440","identity":"rs-1668440","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}