{
  "abstract": "Background Patients with acute myeloid leukemia (AML) are often older, which brings challenges of endurance and persistent efficacy of autologous chimeric antigen receptor (CAR)-T cell therapies. Allogenic CAR-natural killer (NK) cell therapies may offer reduced toxicities and enhanced anti-leukemic potential against AML. CD33 CAR-NK cells have been investigated for AML therapy. However, the fratricide-mediated lysis of CD33-expressing NK cells by CD33 CAR-NK cells limits the expansion and efficacy of CD33 CAR-NK cells. Mesothelin (MSLN), a tumor differentiation antigen, is highly expressed in a fraction of patients with AML, making it a promising target for AML therapy.Methods We designed a novel CD33-MSLN Loop CAR (Loop CAR) and evaluated its antitumor efficacy in human umbilical cord blood-derived NK (UCB-NK) cells and human pluripotent stem cell-derived NK (hPSC-iNK) cells. To further avoid fratricide caused by endogenous CD33 expression in NK cells, we established an hPSC-derived cell line via knockout of the CD33 gene (CD33KO) and engineered Loop CAR. We generated CD33KO-Loop CAR-iNK cells using an organoid induction approach. The efficacy of CD33KO-Loop CAR-iNK cells against tumor cells expressing CD33 and MSLN was investigated both in vitro and in AML xenograft mice.Results Loop CAR-NK cells exhibited superior cytotoxicity against dual-antigen-positive tumor cell lines and primary AML cells compared with CD33 CAR-NK and MSLN CAR-NK cells. Moreover, Loop CAR-NK cells showed upregulated signaling pathways related to NK cell activation and cytotoxic function. The loss of CD33 in iNK cells effectively avoided fratricide, improved expansion ability, and significantly enhanced CD33 and MSLN-mediated specific cytotoxicity of Loop CAR-iNK cells. Moreover, the CD33 KO-Loop CAR-iNK cells demonstrated superior tumor-killing activity in AML xenograft mouse models and significantly prolonged mouse survival.Conclusion Loop CAR empowered both UCB-NK cells and hPSC-iNK cells with superior cytotoxicity against CD33 +MSLN+ tumor cells. Genetic disruption of CD33 avoided fratricide and improved efficacy of Loop CAR-iNK cells against AML. This innovative strategy possesses unique advantages and translational potential for treating AML.",
  "authors": [
    {
      "affiliations": [
        "Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China",
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Yao Wang"
    },
    {
      "affiliations": [
        "Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China",
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China",
        "University of Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Xiujuan Zheng"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China",
        "University of Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Zhiqian Wang"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China",
        "University of Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Ziyun Xiao"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China",
        "University of Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Yunqing Lin"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China",
        "University of Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Fan Zhang"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Yanhong Liu"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China",
        "University of Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Pengcheng Liu"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Qitong Weng"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China",
        "University of Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Leqiang Zhang"
    },
    {
      "affiliations": [
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Chengxiang Xia"
    },
    {
      "affiliations": [
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Dehao Huang"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Lijuan Liu"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Yanping Zhu"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Qi Zhang"
    },
    {
      "affiliations": [
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Hanmeng Qi"
    },
    {
      "affiliations": [
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Yi Chen"
    },
    {
      "affiliations": [
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Yiyuan Shen"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China",
        "University of Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Chenyuan Zhang"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China",
        "University of Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Jiacheng Xu"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China",
        "University of Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Yaoqin Zhao"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China",
        "University of Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Jiaxin Wu"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Tongjie Wang"
    },
    {
      "affiliations": [
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Mengyun Zhang"
    },
    {
      "affiliations": [
        "Department of Hematology, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China"
      ],
      "name": "Minming Li"
    },
    {
      "affiliations": [
        "Department of Hematology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China"
      ],
      "name": "Wenbin Qian"
    },
    {
      "affiliations": [
        "Department of Hematology, Tongji Hospital of Tongji University, Shanghai, China"
      ],
      "name": "Aibin Liang"
    },
    {
      "affiliations": [
        "Department of Hematology, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China"
      ],
      "name": "Xin Du"
    },
    {
      "affiliations": [
        "State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China"
      ],
      "name": "Wenyu Yang"
    },
    {
      "affiliations": [
        "State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China"
      ],
      "name": "Tianyuan Hu"
    },
    {
      "affiliations": [
        "Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China"
      ],
      "name": "Qi Chen"
    },
    {
      "affiliations": [
        "State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China"
      ],
      "name": "Xiaofan Zhu"
    },
    {
      "affiliations": [
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China"
      ],
      "name": "Fangxiao Hu"
    },
    {
      "affiliations": [
        "Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China",
        "State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China",
        "Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China",
        "University of Chinese Academy of Sciences, Beijing, China"
      ],
      "name": "Jinyong Wang"
    }
  ],
  "title": "CD33KO-CD33-mesothelin Loop CAR design avoids fratricide and improves efficacy of iNK cells against acute myeloid leukemia",
  "uid": "fde0a376-ee2c-58c7-9117-2e9986929c2b"
}