CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia

• Published in: Journal of translational medicine 2024-03, Vol.22 (1), p.274-15, Article 274
• Main Authors: Yang, Na, Zhang, Caili, Zhang, Yingchun, Fan, Yuting, Zhang, Jing, Lin, Xiaojin, Guo, Ting, Gu, Yangzuo, Wu, Jieheng, Gao, Jianmei, Zhao, Xing, He, Zhixu
• Format: Article
• Language: English
• Subjects: Acute lymphoblastic leukemia; Acute lymphocytic leukemia; Animals; Antigens; Antigens, CD19 - genetics; Antigens, CD19 - metabolism; CD19 antigen; CD20 antigen; CD69 antigen; Cell Line, Tumor; Cell therapy; Cells; Chimeric antigen receptor; Chimeric antigen receptors; Cloning; Cord blood; Cytotoxicity; Cytotoxicity, Immunologic - genetics; Diseases; Down-regulation; Dual targets; Electroporation; Flow cytometry; Graft versus host disease; Immunotherapy, Adoptive; Killer Cells, Natural; Leukemia; Lymphatic leukemia; Lymphocytes; Lymphocytes T; Lymphoma; Medical prognosis; Mice; mRNA; Natural killer cells; Perforin; Precursor Cell Lymphoblastic Leukemia-Lymphoma - metabolism; Quantitative analysis; Receptors, Chimeric Antigen - metabolism; Relapse; Remission (Medicine); RNA; RNA polymerase; RNA, Messenger - metabolism; Tumor cell lines; Tumors; Umbilical cord; γ-Interferon
• ISSN: 1479-5876; 1479-5876
• DOI: 10.1186/s12967-024-04990-6

Chimeric antigen receptor natural killer (CAR-NK) cells represent a promising advancement in CAR cell therapy, addressing limitations observed in CAR-T cell therapy. However, our prior study revealed challenges in CAR-NK cells targeting CD19 antigens, as they failed to eliminate CD19 Raji cells in NSG tumor-bearing mice, noting down-regulation or loss of CD19 antigen expression in some Raji cells. In response, this study aims to enhance CD19 CAR-NK cell efficacy and mitigate the risk of tumor recurrence due to target antigen escape by developing CD19 and CD20 (CD19/CD20) dual-targeted CAR-NK cells. Initially, mRNA encoding anti-CD19 CARs (FMC63 scFv-CD8α-4-1BB-CD3ζ) and anti-CD20 CARs (LEU16 scFv-CD8α-4-1BB-CD3ζ) was constructed via in vitro transcription. Subsequently, CD19/CD20 dual-targeted CAR-NK cells were generated through simultaneous electrotransfection of CD19/CD20 CAR mRNA into umbilical cord blood-derived NK cells (UCB-NK). Following co-electroporation, the percentage of dual-CAR expression on NK cells was 86.4% ± 1.83%, as determined by flow cytometry. CAR expression was detectable at 8 h post-electric transfer, peaked at 24 h, and remained detectable at 96 h. CD19/CD20 dual-targeted CAR-NK cells exhibited increased specific cytotoxicity against acute lymphoblastic leukemia (ALL) cell lines (BALL-1: CD19 CD20 , REH: CD19 CD20 , Jurkat: CD19 CD20 ) compared to UCB-NK, CD19 CAR-NK, and CD20 CAR-NK cells. Moreover, CD19/CD20 dual-targeted CAR-NK cells released elevated levels of perforin, IFN-γ, and IL-15. Multiple activation markers such as CD69 and cytotoxic substances were highly expressed. The creation of CD19/CD20 dual-targeted CAR-NK cells addressed the risk of tumor escape due to antigen heterogeneity in ALL, offering efficient and safe 'off-the-shelf' cell products. These cells demonstrate efficacy in targeting CD20 and/or CD19 antigens in ALL, laying an experimental foundation for their application in ALL treatment.