CD5-directed CAR5/IL-15 NK cells as a therapeutic platform for T-cell malignancies

Background: Engineered NK cells have increasingly emerged as a powerful allogeneic cell therapy platform, with multiple trials demonstrating meaningful anti-tumor responses, durable clinical activity, and an excellent safety profile without GVHD and low cytokine-release toxicity. Their suitability for large-scale manufacturing makes CAR-NK cells particularly attractive for broad clinical use. T-cell lymphomas and leukemias remain difficult to treat, and although CD5 is a widely expressed and clinically actionable target, CAR-T cell development has been limited by fratricide and manufacturing barriers. A CD5-directed NK cell that avoids these limitations is therefore attractive in this setting. Methods: We developed iC9/CAR5/IL-15 NK cells, an off-the-shelf engineered NK-cell therapy targeting CD5⁺ tumors without fratricide or GVHD. The construct incorporates IL-15 to enhance persistence and the inducible caspase 9 (iC9) safety switch for controlled elimination if needed. A panel of CD5-targeting CAR constructs incorporating different intracellular signaling was generated and screened to identify constructs that optimized NK-cell activation, cytotoxicity, cytokine production, metabolic fitness, and functional persistence. These were tested against multiple CD5⁺ T-ALL and T-cell lymphoma models, followed by in vivo evaluation in xenograft systems and safety testing against normal human tissues and long-term culture. Results: Several signaling domains mediated strong anti-tumor activity, each with distinct advantages. CD28- and DAP10-based designs demonstrated the most consistent performance, supporting potent cytotoxicity and enhanced NK-cell activation. After comparing functional reproducibility, including cytotoxicity, cytokine production, proliferation, and serial killing, and considering manufacturing feasibility together with our prior clinical experience using CD28- signaling in CAR-NK products, the CD28-based iC9/CAR5/IL-15 NK construct was selected as the lead therapeutic candidate. In vivo, CD28-based iC9/CAR5/IL-15 NK cells resulted in rapid tumor regression and a significant survival benefit in CD5⁺ T-ALL xenograft models. Safety studies confirmed the absence of off-target cytotoxicity, and prolonged culture showed no autonomous expansion or phenotypic instability. Conclusion: iC9/CAR5/IL-15 NK cells overcome the barriers that have hindered CD5-directed CAR-T cell approaches, offering a scalable off-the-shelf therapy for aggressive T-cell malignancies. A Phase I/II clinical trial evaluating this therapy in relapsed/refractory CD5⁺ T-cell cancers is now open and accruing at our center.