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2B4 co-stimulation and dasatinib modulation enhance anti-CD19 CAR-NK-92 cell cytotoxicity
Matheus Henrique Dos Santos1, Júlia Teixeira Cottas de Azevedo2, Mara Elisama da Silva Januário1
1Center for Cell-based Therapy (CTC), Regional Blood Center of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
Frontiers in Immunology
|December 29, 2025
Summary
Optimizing Chimeric Antigen Receptor (CAR)-NK cells with NK-specific co-stimulatory domains like 2B4 and DAP12 enhances cytotoxicity. Transient dasatinib treatment reversibly modulates CAR-NK function, improving tumor control in vivo.
Area of Science:
- Immunology
- Cell Biology
- Oncology
Background:
- Chimeric Antigen Receptor (CAR)-based therapies have revolutionized cancer treatment, particularly for hematological malignancies.
- While CAR-T cell co-stimulatory domains are well-understood, optimal signaling modules for CAR-Natural Killer (CAR-NK) cells require further definition.
- Tailoring co-stimulatory domains is crucial for enhancing CAR-NK cell-mediated cytotoxicity and therapeutic potential.
Purpose of the Study:
- To engineer and evaluate CAR19 constructs with NK-specific co-stimulatory domains (2B4, DAP12) using the NK-92 cell line.
- To assess the impact of these domains on CAR-NK cell function, including cytotoxicity and cytokine production.
- To investigate the pharmacologic modulation of CAR-NK cells using transient dasatinib treatment and its effects on in vivo antitumor activity.
Main Methods:
- Engineering of CAR19 constructs incorporating 2B4 and DAP12 co-stimulatory domains in the NK-92 cell line.
- Functional assays (cytotoxicity, cytokine production) and transcriptomic profiling to evaluate CAR-NK cell responses.
- In vivo studies using a xenograft model to assess antitumor efficacy of engineered CAR-NK cells, with and without dasatinib pretreatment.
Main Results:
- CAR constructs with 2B4 and 2B4-DAP12 domains significantly enhanced NK cytotoxic programming, confirmed by functional assays and transcriptomic signatures.
- Short-term dasatinib treatment reversibly suppressed CAR-NK effector function, but led to enhanced activity upon drug withdrawal.
- In vivo, 2B4-DAP12 CAR19-NK-92 cells pretreated with dasatinib demonstrated superior tumor control compared to conventional 4-1BBζ CAR19-NK-92 cells.
Conclusions:
- Selection of NK-specific co-stimulatory domains is critical for optimizing CAR-NK cell performance.
- Reversible inhibition of Src-family kinases via dasatinib offers a strategy to enhance CAR-NK therapy efficacy.
- The NK-92 cell line serves as a valuable proof-of-concept platform for dissecting CAR signaling mechanisms and informing the development of next-generation CAR-NK therapies for primary NK cells.
