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

  • J Transl Med. 2024 Mar 13;22(1):274. doi: 10.1186/s12967-024-04990-6.
Na Yang  1  2 ,  Caili Zhang  1 ,  Yingchun Zhang  3 ,  Yuting Fan  1  2 ,  Jing Zhang  3 ,  Xiaojin Lin  1  3 ,  Ting Guo  4 ,  Yangzuo Gu  5 ,  Jieheng Wu  2 ,  Jianmei Gao  6 ,  Xing Zhao  7  8  9 ,  Zhixu He  10  11  12  13
Affiliations
  • 1. Tissue Engineering and Stem Cell Experiment Center, Guizhou Medical University (GMU), Guiyang, Guizhou, China.
  • 2. Department of Immunology, College of Basic Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, China.
  • 3. Department of Biology, School of Basic Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, China.
  • 4. Department of Gynecology, the Affiliated Hospital of Guizhou Medical University, Guiyang, China.
  • 5. State Key Laboratory of Biotherapy and Cancer Center, Sichuan University, Chengdu, China.
  • 6. School of Pharmacy, Zunyi Medical University, Zunyi, China.
  • 7. Tissue Engineering and Stem Cell Experiment Center, Guizhou Medical University (GMU), Guiyang, Guizhou, China. [email protected].
  • 8. Department of Immunology, College of Basic Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, China. [email protected].
  • 9. Department of Biology, School of Basic Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, China. [email protected].
  • 10. Tissue Engineering and Stem Cell Experiment Center, Guizhou Medical University (GMU), Guiyang, Guizhou, China. [email protected].
  • 11. Department of Immunology, College of Basic Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, China. [email protected].
  • 12. Key Laboratory of Adult Stem Cell Translational Research, Chinese Academy of Medical Sciences), Guiyang, China. [email protected].
  • 13. Department of Pediatrics, the Affiliated Hospital of Zunyi Medical University, Zunyi, China. [email protected].
PMID: 38475814 DOI: 10.1186/s12967-024-04990-6
Abstract

Background: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

Keywords
Acute lymphoblastic leukemia; Chimeric antigen receptor; Dual targets; Natural killer cells; mRNA.
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