NK-92MI Cells Engineered with Anti-claudin-6 Chimeric Antigen Receptors in Immunotherapy for Ovarian Cancer

Int J Biol Sci. 2024 Feb 11;20(5):1578-1601. doi: 10.7150/ijbs.88539. eCollection 2024.

Abstract

Background: The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR-NK cell efficacy. Claudin-6 (CLDN6) has been reported to be overexpressed in ovarian cancer and may be an attractive target for CAR-NK cells immunotherapy. However, the feasibility of using anti-CLDN6 CAR-NK cells to treat ovarian cancer remains to be explored. Methods: CLDN6 expression in primary human ovarian cancer, normal tissues and cell lines were detected by immunohistochemistry and western blot. Two types of third-generation CAR NK-92MI cells targeting CLDN6, CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) and CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB) were constructed by lentivirus transfection, sorted by flow cytometry and verified by western blot and qPCR. OVCAR-3, SK-OV-3, A2780, Hey and PC-3 cells expressing the GFP and luciferase genes were transduced. Subcutaneous and intraperitoneal tumor models were established via NSG mice. The ability of CLDN6-CAR NK cells to kill CLDN6-positive ovarian cancer cells were evaluated in vitro and in vivo by live cell imaging and bioluminescence imaging. Results: Both CLDN6-CAR1 and CLDN6-CAR2 NK-92MI cells could specifically killed CLDN6-positive ovarian cancer cells (OVCAR-3, SK-OV-3, A2780 and Hey), rather than CLDN6 negative cell (PC-3), in vitro. CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) exhibited stronger cytotoxicity than CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB). Furthermore, CLDN6-CAR1 NK cells could effectively eliminate ovarian cancer cells in subcutaneous and intraperitoneal tumor models. More importantly, CAR-NK cells combined with immune checkpoint inhibitors, anti-PD-L1, could synergistically enhance the antitumor efficacy of CLDN6-targeted CAR-NK cells. Conclusions: These results indicate that CLDN6-CAR NK cells possess strong antitumor activity and represent a promising immunotherapeutic modality for ovarian cancer.

Keywords: Claudin-6; NK cells; PD-1; PD-L1; chimeric antigen receptor; ovarian cancer.

MeSH terms

  • Animals
  • Apoptosis
  • CD28 Antigens / metabolism
  • Cell Line, Tumor
  • Claudins*
  • Female
  • Humans
  • Immunotherapy / methods
  • Immunotherapy, Adoptive / methods
  • Killer Cells, Natural
  • Mice
  • NK Cell Lectin-Like Receptor Subfamily K / metabolism
  • Ovarian Neoplasms* / metabolism
  • Ovarian Neoplasms* / therapy
  • Receptors, Chimeric Antigen* / genetics

Substances

  • Receptors, Chimeric Antigen
  • claudin 6
  • NK Cell Lectin-Like Receptor Subfamily K
  • CD28 Antigens
  • Claudins