O-10.01 CD44v6-specific CAR-NK cells: a novel approach to precision medicine in bladder cancer

<h3>Background</h3> Limited advances in bladder cancer (BC) management represent a major health and social problem. Specifically, the combination of periodic revisions using invasive techniques and the current aggressive therapies reduces the quality-of-life of BC patients significantly. Moreover, at present, there are no stratification strategies for precision medicine-based therapeutic decisions. Recently, we characterized CD44v6 as a novel tumor associated antigen in BC, representing a potential predictive and prognostic biomarker as well as therapeutic target for directed therapy in BC. Accordingly, the objective of this study is the development of CD44v6-directed BC therapy (CD44v6-specific CAR-NK cells), based on a single-chain variable fragment (scFv) antibody against CD44v6. <h3>Materials and Methods</h3> Employing a comprehensive methodological approach, including real-time interaction monitoring as well as <i>in vitro</i> and <i>in vivo</i> specificity and cytotoxicity assays, we evaluated the kinetic characteristics and cytotoxic potential of newly engineered CD44v6-specific antibody fragments and CD44v6-specific CAR-NK cells. For all <i>in vivo</i> assays, orthotopic xenograft mouse models (human BC cell injection in the murine bladder wall) were used. <i>In vivo</i> imaging was performed using a combination of bioluminescent and fluorescent imaging. <h3>Results</h3> We successfully expressed and purified the recombinant scFv CD44v6 antibody. Using LigandTracer technology, we monitored the real-time interaction between the scFv CD44v6 antibody and CD44v6 receptor on various BC cell lines. The interaction followed a one-to-one model, yielding an apparent affinity (K_D) of ±1 nM, an association rate constant (Ka) of ±1.69 × 10⁴ M⁻¹s⁻¹, and a dissociation rate constant (Kd) of ±2.04 × 10⁻⁵ s⁻¹. Specificity assays further confirmed the specific binding of scFv CD44v6 to the CD44v6 receptor. <i>In vivo</i> imaging revealed specific tumor targeting by the scFv CD44v6, without off-site binding, in an orthotopic mouse model. Using this extensively evaluated scFv CD44v6 antibody as CAR, we generated functional CD44v6-specific CAR-NK cells. Real-time monitoring of CAR-NK cell interactions with BC cells confirmed the interaction of CD44v6-specific CAR-NK cells, but not NK-92 cells, with the CD44v6 receptor on multiple BC cell lines. <i>In vitro</i> cytotoxicity assays showed that CD44v6-specific CAR-NK cells had enhanced cytotoxic activity against BC cells compared to NK-92 cells, with a positive correlation between cytotoxicity and CD44v6 receptor density. <i>In vivo</i> imaging revealed specific tumor targeting by the CD44v6-specific CAR-NK cells in an orthotopic mouse model. <h3>Conclusions</h3> We generated CD44v6-specific CAR-NK cells which show high affinity binding, specific CD44v6 receptor targeting and cytotoxic activity against CD44v6-expressing tumor cells. Our findings highlight the potential of CD44v6-specific CAR-NK cells as innovative therapeutic agents for more effective and tailored approaches to combat BC disease. I. Lodewijk: None. L. Morales: None. M. Turon-Orra: None. E. Montesinos: None. C. Suárez-Cabrera: None. V.G. Martínez: None. E. Castellano: None. A. Valeri: None. J. Nilvebrant: None. M. Nestor: None. J.M. Paramio: None. M. Dueñas: None.