GD2-TARGETING CAR-NK CELLS ENHANCED BY TRANSGENIC GITRL EXPRESSION ARE AN EFFECTIVE FOR GLIOBLASTOMA AND MELANOMA

Background & Aim Allogeneic chimeric antigen receptors (CAR) NK therapies offer a solution to certain limitations of autologous CAR-T therapy, as NK cells are not constrained by patient compatibility issues and can be sourced from healthy donors for off-the-shelf applications. Also, their presence in the tumor microenvironment is associated with favorable prognoses. Treating solid tumors poses an additional challenge due to their highly immunosuppressive environment, intensified, in part, by Tregs. However, Tregs have inactivation receptors, such as GITR, which has its action blocked when it interacts with its ligand. Neuroectodermal tumors, such as glioblastomas and melanomas, typically exhibit an overexpression of the GD2 tumor antigen. This study aims to produce, characterize, and evaluate the therapeutic potential of anti-GD2 CAR-NK cells co-expressing GITRL, aiming for greater response within the tumor microenvironment by blocking Tregs. CAR.GD2 and CAR.GD2.GITRL were both characterized. In vitro cytotoxicity assays were conducted by flow cytometry and bioluminescence methods. Methods, Results & Conclusion Both CAR cells achieved a robust 90% CAR expression post-positive selection, maintaining stability even after cryopreservation. This resilience in CAR expression suggests a potential use as an off-the-shelf therapy. CAR.GD2.GITRL showed 47.8% of GITRL expression. Moreover, CAR-NK cells demonstrated a superior expansion factor and growth rate compared to NK-92. GD2 tumor cell lines from melanoma SK-MEL28-S6, glioblastoma U251 and GD2 negative tumor cell line HCT116 were used in cytotoxicity assays at a 1:2 (effector: target) ratio for 12 and 48 hours. NK-92 cells were less cytotoxic than CARs across all groups. CAR.GD2 demonstrated cytotoxicity exceeding 45% after 12 hours, and 87% after 48 hours in both cell lines. CAR.GD2.GITRL exhibited superior cytotoxicity, surpassing 57% after 12 hours and 99% after 48 hours in both tumor cells, as confirmed by degranulation assays. These results highlight enhanced cytotoxicity against GD2+ cells. Notably, CAR.GD2.GITRL demonstrated a higher anti-tumor capacity than CAR.GD2, suggesting a potential co-stimulatory role of GITRL. In the next phases, we will investigate the impact of our CARs on primary glioblastoma and melanoma cells, and assess their efficacy in vivo models. Financial support: Fapesp 2021/10530-3, 2020/07055-9, 2014/50947-7, 2013/08135-2, CNPq 440543/2022-3. Allogeneic chimeric antigen receptors (CAR) NK therapies offer a solution to certain limitations of autologous CAR-T therapy, as NK cells are not constrained by patient compatibility issues and can be sourced from healthy donors for off-the-shelf applications. Also, their presence in the tumor microenvironment is associated with favorable prognoses. Treating solid tumors poses an additional challenge due to their highly immunosuppressive environment, intensified, in part, by Tregs. However, Tregs have inactivation receptors, such as GITR, which has its action blocked when it interacts with its ligand. Neuroectodermal tumors, such as glioblastomas and melanomas, typically exhibit an overexpression of the GD2 tumor antigen. This study aims to produce, characterize, and evaluate the therapeutic potential of anti-GD2 CAR-NK cells co-expressing GITRL, aiming for greater response within the tumor microenvironment by blocking Tregs. CAR.GD2 and CAR.GD2.GITRL were both characterized. In vitro cytotoxicity assays were conducted by flow cytometry and bioluminescence methods. Both CAR cells achieved a robust 90% CAR expression post-positive selection, maintaining stability even after cryopreservation. This resilience in CAR expression suggests a potential use as an off-the-shelf therapy. CAR.GD2.GITRL showed 47.8% of GITRL expression. Moreover, CAR-NK cells demonstrated a superior expansion factor and growth rate compared to NK-92. GD2 tumor cell lines from melanoma SK-MEL28-S6, glioblastoma U251 and GD2 negative tumor cell line HCT116 were used in cytotoxicity assays at a 1:2 (effector: target) ratio for 12 and 48 hours. NK-92 cells were less cytotoxic than CARs across all groups. CAR.GD2 demonstrated cytotoxicity exceeding 45% after 12 hours, and 87% after 48 hours in both cell lines. CAR.GD2.GITRL exhibited superior cytotoxicity, surpassing 57% after 12 hours and 99% after 48 hours in both tumor cells, as confirmed by degranulation assays. These results highlight enhanced cytotoxicity against GD2+ cells. Notably, CAR.GD2.GITRL demonstrated a higher anti-tumor capacity than CAR.GD2, suggesting a potential co-stimulatory role of GITRL. In the next phases, we will investigate the impact of our CARs on primary glioblastoma and melanoma cells, and assess their efficacy in vivo models. Financial support: Fapesp 2021/10530-3, 2020/07055-9, 2014/50947-7, 2013/08135-2, CNPq 440543/2022-3.