Development of anti-CD19-CAR specific for NK cells to enhance antitumoral effects for the treatment of leukemia and lymphoma.

Abstract

Hematological malignancies represent a significant portion among the diagnoses of neoplastic cells. Adoptive cell therapy with T cells genetically modified to express chimeric antigen receptors(CAR) has revolutionized the field, with promising and impressive results. However, there are still obstacles associated with the use of CAR-modified T cells, such as the need of autologous cells and cytotoxicity related to the cytokines, making the prospection of new therapeutic alternatives to be encouraged. NK cells have become an attractive alternative for the application of CAR technology, since these cells have a fundamental role in immunity against tumors, so, its cytotoxic activity can be specifically redirected to the target through modification with CAR. The main advantage of this cells is safeguarded in the use of allogenic cell sources, since they have the potential to not induce or minimally induce graft versus host disease. An important field in CAR-NK context is the constructions of new CARs carrying structures derived from native NK molecules, since it was found that the use of conventional CARs originally designed for T cells may not trigger robust effectors signals in NK cells. Recently, our research group successfully developed four anti-CD19 CARs containing combinations of signaling regions derived from molecules 4-1BB, 2B4, DAP12 and CD3¶ in order to obtain better effector responses from NK cells. To contribute to the advancement of the above topic, the current proposal has the aim to comparatively deepen the characterization of the new constructions in NK-92 cells at the level of the signaling cascades recruited by the different CARs through the phosphoflow technique in flow cytometry. Subsequently, we will validate the effect of the best constructs in vivo. In addition, the present research project proposes the creation of new CARs constructs based on the stalk and transmembrane regions of the NKp30 and NKp46 molecules and in association with the complete sequence of the CD3¶ molecule, aiming to recapitulate the interaction that these molecules present in the context native activation of NK cells and, thus, trigger better cytotoxic effects directed to CD19 positive cells. Therefore, the results of this project have the potential to contribute to the generation of NK-CAR cells with greater activation capacity and cytotoxic activity.