P-08.01 A combinatorial therapy for bladder cancer: epigenetic inhibitor and CAR-NK cells

<h3>Background</h3> Bladder cancer (BC) is among the ten most common types of cancer and causes around 220,000 deaths per year worldwide since current therapies cannot be used in a large proportion of patients due to comorbidities. Recently, new treatments have been developed, including immunotherapies such as immune checkpoint inhibitors or antibody drug conjugates. However, since those treatments are only effective in a small subset of patients, development of new, more effective, and less invasive therapies is needed. Natural Killer cells expressing chimeric antigen receptors (CAR-NKs) are currently being tested as a promising cell-based immunotherapy against different solid tumors. On the other hand, epigenetic modifications are drivers of BC and their modulation by inhibitors is considered a promising approach for BC management. These epigenetic inhibitors also show immunomodulatory properties, including NK cells` antitumor activities. Here, we explore an advanced cell immunotherapy combined with an epigenetic inhibitor as a therapeutic strategy for advanced BC patients. <h3>Materials and Methods</h3> Several human BC cell lines were treated with a novel inhibitor of epigenetic machinery (CM-1758) against histone deacetylases. The induction of the NK cell ligands (NKG2DLs) surface expression by CM-1758 was checked by flow cytometry. NK-92 MI cells that express high levels of CAR, comprising a fusion of NKG2D receptor ectodomain with 4-1BB and CD3ζ, were co-cultured with BC cells previously treated or not with CM-1758 to measure NKG2D CAR-NK-92 MI cytotoxicity. We set up an orthotopic mouse model by intramural injection in the bladder of RT112 cells expressing Firefly luciferase to follow up tumor growth in nude mice. Additionally, we generated NKG2D CAR-NK-92 MI cells that express Cypridina luciferase to analyze their biodistribution, persistence and tumor targeting ability in our mouse models. Currently, we are optimizing the dose schedule for the combinatorial therapy using CM-1758 epigenetic inhibitor and NKG2D CAR-NK-92 MI cells <i>in vivo</i>. <h3>Results</h3> CM-1758 led to growth inhibition in a variety of human BC cell lines (5637, RT112, 253J, J82 and TCCSUP) in the low micromolar range. Moreover, CM-1758 induced the NKG2DL expression in four of these BC cell lines. We observed a potent cytotoxic effect of NKG2D CAR-NK-92 MI cells in co-culture with all BC cell lines tested <i>in vitro</i>. Likewise, cells treated with CM-1758 increased their sensitivity to NKG2D CAR-NK-92 MI cells in co-culture experiments and this effect was dependent on NKG2D receptor. The biodistribution study of NK-92 MI cells revealed that these cells persist in nude mice for at least seven days post-administration. Importantly, the levels of NKG2D CAR-NK-92 MI cells were higher in tumors in comparison with normal bladder and this effect was greater for NKG2D CAR-NK-92 MI cells as compared with the parental NK-92 MI cell line. <h3>Conclusions</h3> Our results support that treatment with epigenetic inhibitors may facilitate the use of CAR-NK-based cell therapies as a promising strategy for BC management. The orthotopic murine model of BC that has been established using human RT112 cells is useful to test CAR-NK therapy. In future preclinical studies, we hope to evaluate the effect of the combinatorial CM-1758 and NKG2D CAR-NK-92 MI <i>in vivo</i>. L. Morales: None. M. Turon-Orra: None. S.P. Nunes: None. I. Lodewijk: None. M. Perez-Escavy: None. C. Suárez-Cabrera: None. J.A. Casado: None. P. Río: None. A. Valeri: None. E. San José-Enériz: None. X. Agirre: None. F. Prosper: None. J.M. Paramio: None.