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A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells
Published on: November 12, 2019
Dual CAR-NK cells targeting PD-L1 and ErbB2 (HER2) exhibit cooperative CAR signaling and counteract solid tumor
Kristin Freudenberg-Jahn1,2, Idan Ben-Horin3, Nivedha Murali Shankar1,2
1Experimental Transfusion Medicine, Faculty of Medicine Carl Gustav Carus, Dresden University of Technology, Dresden, Germany.
Background:
Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown potent efficacy against hematologic malignancies. In contrast, solid tumors remain difficult to treat due to heterogeneous antigen expression and an immunosuppressive tumor microenvironment that fosters immune evasion and is driven to a large part by checkpoint-mediated inhibition.
Methods:
To overcome these barriers, we engineered novel dual CAR-NK cells targeting the immune checkpoint PD-L1 and the tumor-associated antigen ErbB2 (HER2). The clinically applicable NK-92 cell line was lentivirally transduced with PD-L1.CAR and ErbB2.CAR constructs. Antitumor activity was assessed across diverse solid tumor models using a comprehensive panel of in vitro and in vivo assays.
Results:
Dual CAR-NK cells exhibited potent and selective cytotoxicity against breast, pancreatic, gastric, and lung cancer cell lines expressing either one or both targets. Comparable efficacy was observed in 3D spheroid cultures and against patient-derived primary ovarian cancer cells. Notably, dual CAR-NK cells retained strong cytotoxicity when one antigen was absent or blocked, modeling immune escape through antigen loss - a feature absent in single CAR-NK cells. Mechanistically, each CAR independently activated the MEK/ERK pathway in an antigen-dependent manner, while dual CAR stimulation potentiated the signaling response in a cooperative manner. Furthermore, IFN-γ produced by NK cells upon encounter of resistant cancer cells was sufficient to induce PD-L1 upregulation, thereby enhancing the potency of the dual CAR system. In a PD-L1/ErbB2 double-positive breast cancer xenograft model, dual CAR-NK cells consistently outperformed single-target controls.
Conclusions:
Dual targeting of PD-L1 and ErbB2 enhances CAR-NK cell efficacy against refractory solid tumors by providing resilience to antigen heterogeneity and amplifying antitumor signaling through cooperative activation. This approach represents a promising and adaptable platform for clinical translation in solid tumor immunotherapy.
Insights
Engineered dual Chimeric Antigen Receptor (CAR)-Natural Killer (NK) cells targeting PD-L1 and ErbB2 show enhanced efficacy against solid tumors. This dual-targeting approach overcomes antigen loss and immunosuppression for improved cancer immunotherapy.
Area of Science:
- Immunology
- Cancer Biology
- Cell Therapy
Background:
- Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells are effective against blood cancers but struggle with solid tumors due to antigen heterogeneity and immunosuppressive microenvironments.
- Immune evasion by solid tumors is often driven by checkpoint-mediated inhibition, hindering the effectiveness of single-target immunotherapies.
Purpose of the Study:
- To engineer novel dual CAR-NK cells targeting both the immune checkpoint PD-L1 and the tumor-associated antigen ErbB2 (HER2).
- To evaluate the efficacy and mechanisms of these dual CAR-NK cells against various solid tumor models.
Main Methods:
- Lentiviral transduction of the NK-92 cell line with PD-L1.CAR and ErbB2.CAR constructs.
- Assessment of antitumor activity using in vitro assays (cell lines, 3D spheroids, patient-derived cells) and in vivo xenograft models.
- Mechanistic studies involving signaling pathway analysis (MEK/ERK) and cytokine production (IFN-γ).
Main Results:
- Dual CAR-NK cells demonstrated potent and selective cytotoxicity against breast, pancreatic, gastric, and lung cancer cell lines expressing either or both targets.
- The engineered cells maintained efficacy even when one target antigen was absent or blocked, addressing antigen loss immune escape.
- Dual CAR stimulation cooperatively enhanced MEK/ERK signaling, and NK cell-produced IFN-γ upregulated PD-L1, further boosting efficacy.
- In vivo studies showed dual CAR-NK cells outperformed single-target CAR-NK cells in a relevant breast cancer model.
Conclusions:
- Dual targeting of PD-L1 and ErbB2 significantly enhances CAR-NK cell efficacy against refractory solid tumors.
- This strategy provides resilience to antigen heterogeneity and amplifies antitumor signaling, offering a promising platform for solid tumor immunotherapy.
- The adaptable dual CAR-NK cell platform holds potential for clinical translation in treating challenging solid tumors.
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