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Updated: Jul 10, 2026
Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells
Published on: February 14, 2025
Engineered PD1-NKG2D Dual-CAR NK92 cells broaden antitumor target recognition in preclinical tumor models
Xinru Jin1,2, Mingfeng Li1, Mengjun Wang1
1Henan Province Engineering Technology Research Center of Advanced Synbiomedicine and Clinical Innovation Translation, School of Life Sciences and Technology, Henan Medical University, Xinxiang, Henan, China.
Background:
To address challenges such as the complex manufacturing of CAR-T and the immunosuppressive tumor microenvironment (TME), CAR-NK cells offer greater potential as an "off-the-shelf" therapy. To broaden tumor recognition and reduce the risk of immune escape associated with single-target approaches, we developed a single-promoter-driven multicistronic CAR-NK92 system that employs NKG2D for broad recognition of stress ligands and combines a PD1-CAR to reverse PD-L1 inhibitory signaling, thereby significantly enhancing antitumor efficacy.
Methods:
A multicistronic construct co-expressing PD1-CAR and NKG2D-CAR was generated using a P2A peptide under the control of a single CMV promoter and introduced into NK92 cells. The expression of PD-L1 and MICA/B was screened across multiple tumor cell lines, and the functional robustness of PN-CAR-NK92 cells was evaluated in all models through in vitro cytotoxicity and cytokine secretion assays. The in vivo translational efficacy was further validated using an H1299 xenograft model, with a direct comparison between PN-CAR-NK92 cells and NK92 cells.
Results:
The multicistronic design enabled stable surface co-expression of both receptor modules, providing a structural basis for dual-target functionality. In vitro cytotoxicity assays demonstrated that PN-CAR-NK92 cells maintained robust antitumor activity across tumor cell lines with distinct PD-L1 and MICA/B expression profiles, whereas single-target CAR-NK92 cells displayed more restricted target specificity. These findings suggest that dual-target CAR engineering broadens antigen recognition coverage and may help reduce the limitations associated with single-target antigen dependence. Furthermore, PN-CAR-NK92 cells demonstrated significantly enhanced tumor suppression in the H1299 xenograft model compared with control groups.
Conclusions:
Dual-target PD1/NKG2D CAR-NK92 cells exhibit broadened antitumor activity across tumor cells with distinct ligand-expression profiles and may represent a promising strategy to reduce the limitations associated with single-target CAR therapies.
Insights
Engineered CAR-NK92 cells with dual PD1 and NKG2D receptors show enhanced antitumor activity against diverse tumors. This dual-targeting approach broadens cancer cell recognition and overcomes single-target limitations for improved efficacy.
Area of Science:
- Immunotherapy
- Cellular Engineering
- Oncology
Background:
- CAR-T cell manufacturing complexity and tumor microenvironment (TME) challenges necessitate alternative therapies.
- CAR-NK cells present an "off-the-shelf" immunotherapy option with broader potential.
- Single-target CAR approaches risk immune escape; dual-targeting aims to enhance tumor recognition and efficacy.
Purpose of the Study:
- To develop a single-promoter-driven multicistronic CAR-NK92 system for enhanced antitumor efficacy.
- To broaden tumor recognition by combining NKG2D for stress ligand recognition and PD1-CAR to counter PD-L1 signaling.
- To overcome limitations of single-target CAR therapies by engineering dual-target CAR-NK92 cells.
Main Methods:
- Generated a multicistronic construct co-expressing PD1-CAR and NKG2D-CAR using a P2A peptide under a single CMV promoter.
- Introduced the construct into NK92 cells, creating PN-CAR-NK92 cells.
- Evaluated in vitro cytotoxicity and cytokine secretion against tumor cell lines expressing PD-L1 and MICA/B; validated in vivo efficacy using an H1299 xenograft model.
Main Results:
- Stable surface co-expression of both PD1-CAR and NKG2D-CAR modules was achieved in PN-CAR-NK92 cells.
- PN-CAR-NK92 cells demonstrated robust antitumor activity across diverse tumor cell lines, outperforming single-target CAR-NK92 cells with restricted specificity.
- Significantly enhanced tumor suppression was observed in the H1299 xenograft model compared to control groups.
Conclusions:
- Dual-target PD1/NKG2D CAR-NK92 cells exhibit broadened antitumor activity against tumors with varied ligand expression profiles.
- This dual-targeting strategy effectively overcomes limitations associated with single-target CAR therapies.
- PD1/NKG2D CAR-NK92 cells represent a promising advancement in CAR-NK cell-based cancer immunotherapy.
