Hypoimmunogenic CD19 CAR-NK cells derived from embryonic stem cells suppress the progression of human B-cell malignancies in xenograft animals

Front Immunol. 2024 Nov 27:15:1504459. doi: 10.3389/fimmu.2024.1504459. eCollection 2024.

Abstract

Background: Chimeric antigen receptor (CAR) engineered natural killer (NK) cells exhibit advantages such as MHC-independent recognition and strong anti-tumor functions. However, allogeneic CAR-NK cells derived from human tissues are heterogeneous and susceptible to clearance by hosts.

Methods: We generated a B2M knockout, HLA-E and CD19 CAR ectopic expressing embryonic stem cell (ESC) line, which differentiated normally and gave rise to homogeneous CD19 CAR-NK (CD19 CAR-UiNK) cells using an organoid aggregate induction method. The CD19 CAR-UiNK were co-cultured with T cells or NK cells derived from peripheral blood mononuclear cells (PBMC) with the mismatched HLA to evaluate the immunogenicity of CD19 CAR-UiNK cells. We further assessed the therapeutic effects of CD19 CAR-UiNK cells on CD19+ tumor cells through in vitro cytotoxicity assays and in vivo animal models.

Results: The CD19 CAR-UiNK cells exhibited typical expression patterns of activating and inhibitory receptors, and crucial effector molecules of NK cells, similar to those of unmodified NK cells. In co-culture assays, the CD19 CAR-UiNK cells evaded allogeneic T cell response and suppressed allogeneic NK cell response. Functionally, the CD19 CAR-UiNK cells robustly secreted IFN-γ and TNF-α, and upregulated CD107a upon stimulation with Nalm-6 tumor cells. The CD19 CAR-UiNK cells effectively eliminated CD19+ tumor cells in vitro, including B-cell cancer cell lines and primary tumor cells from human B-cell leukemia and lymphoma. Further, the CD19 CAR-UiNK cells exhibited strong anti-tumor activity in xenograft animals.

Conclusion: We offer a strategy for deriving homogeneous and hypoimmunogenic CD19 CAR-iNK cells with robust anti-tumor effects from ESCs. Our study has significant implications for developing hypoimmunogenic CD19 CAR-NK cell therapy using human ESC as an unlimited cell source.

Keywords: B-cell malignancy; CD19 CAR-NK cells; cytotoxicity; embryonic stem cells; hypoimmunogenicity.

MeSH terms

  • Animals
  • Antigens, CD19* / immunology
  • Cell Line, Tumor
  • Cytotoxicity, Immunologic
  • Embryonic Stem Cells* / immunology
  • Humans
  • Immunotherapy, Adoptive / methods
  • Killer Cells, Natural* / immunology
  • Leukemia, B-Cell / immunology
  • Leukemia, B-Cell / therapy
  • Lymphoma, B-Cell / immunology
  • Lymphoma, B-Cell / therapy
  • Mice
  • Receptors, Chimeric Antigen* / genetics
  • Receptors, Chimeric Antigen* / immunology
  • Receptors, Chimeric Antigen* / metabolism
  • Xenograft Model Antitumor Assays*

Substances

  • Antigens, CD19
  • Receptors, Chimeric Antigen

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by grants from the National Natural Science Foundation of China (81925002, 82100128, 82300132, 32300676) and the National Key R&D Program of China (2020YFA0112404).