Evaluation of GITRL coexpression on the therapeutic efficiency and immunossupression resistance of anti-GD2 CAR-T cells in a preclinical model of Glioblastoma Multiforme

Abstract

The cancer immunotherapies have shown promising results compared to the conventional therapies, such as the adoptive therapy of T lymphocytes expressing chimeric antigen receptors (CAR) in the treatment of hematological malignancies. However, the therapeutic efficacy of CAR-T lymphocytes against solid tumors, which represent the majority of tumors, is still insufficient. One of the reasons for this scenario is attributed to the tumor microenvironment, composed of a series of extracellular matrix proteins, cells and molecules with immunosuppressive capacity that mitigate the infiltration of CAR-T cells and attenuate their cytolytic activity. The presence of these immunosuppressive agents subverts the response against tumors in the immune escape mechanisms, leading to the immunosuppression and modest therapeutic efficiency. Therefore, it is essential to improve the biotechnologies used in the development of CAR-T lymphocytes in order to enhance the action of these cells and to overcome immunosuppressive barriers. The functionality of T cells is directly related to activation, which occurs through a sequence of signals from antigenic recognition via TCR followed by engagement between the costimulatory molecules. In this context, an important molecule for the consolidation of the T cell activation is the glucocorticoid-induced TNF receptor-related protein (GITR). The activation of GITR occurs from the interaction with its ligand (GITRL), generating the costimulatory signals on effector T cells and suppression on regulatory T cells, making it a promising immunotherapeutic target. Therefore, the premise of this work is that the incorporation of a GITRL domain in CAR-T lymphocytes will have a combined action of potentiating not only the functionality of these cells, but also suppressing the immunosuppression exerted by regulatory T cells. Thus, this project aims to recreate the architecture and immunosuppressive mechanisms of the tumor microenvironment to assess the therapeutic efficiency and immunosuppression resistance of anti-GD2 CAR-T lymphocytes. It is a highly oncogenic membrane glycosphingolipid expressed in tumors of neuroectodermal origin, such as glioblastoma multiforme. This neoplasm represents the most prevalent and aggressive type of malignant brain tumor, in addition to being known to have a highly immunosuppressive nature. To test our hypothesis, primary culture of human T lymphocytes transduced with lentiviral vectors coding for CAR.GD2 and CAR.GD2/GITRL will be evaluated for their target-specific antineoplastic activity and resistance to immunosuppression both in vitro, by a 3D multicellular spheroid co-culture assay containing tumor and regulatory cells, and in vivo by co-injection of T regulatory cells in a preclinical orthotopic model of human glioblastoma multiforme. From preliminary results, we performed the generation and functional validation in vitro of the two vectors that we used to modify the anti-GD2 CAR-T lymphocytes. In this way, we hope that the incorporation of these analyzes with greater similarity with the tumor microenvironment will provide the improvement of the success of this immunotherapy against solid tumors. The development and validation of this construction in primary human T lymphocytes will be an important opportunity to expand treatment possibilities within the scope of the Cell Therapy Center (CEPID project 013/08135-2) with the inclusion of GD2+ neoplasms. The potential findings of this work will contribute to the expansion of the advanced cell therapies and the consolidation of the use of CAR-T cells as a truly disruptive therapeutic approach in the treatment of hitherto incurable neoplasms. (AU)