Controlling infection using NK cells expressing a Chimeric Antigen Receptor (CAR) specific to Cryptococcus spp

Abstract

Chimeric Antigen Receptor (CAR) therapy is a new approach that utilizes bioengineering to modify immune cells so they are able to specifically target and kill pathogens or altered host immune cells (such as cancers). This approach involves generating modified cell surface molecules (chimeric receptors) and expressing these receptors in host immune cells. As these chimeric receptors recognize a desired target, expression of these receptors enables the modified host cells to specifically kill the invading pathogen or cancerous cell. In a project funded by FAPESP, the da Silva's laboratory has evaluated the capacity of CAR to redirect T and NK cells to target invasive fungal infections. This research group has developed CAR constructs specific to Cryptococcus spp using monoclonal antibodies (18B7 and 2H1 clone) that are able to recognize GXM, called GXMR-CAR. This receptor expressed by human T cells mediated a reduction of titan cells of C. neoformans in the lungs of infected animals (NSG mice). C. neoformans commonly affects immunocompromised patients, such as individuals with HIV or those who have undergone to the treatment with immunosuppressive drugs; whereas C. gattii is considered a primary pathogen because of its ability to infect healthy individuals. Cryptococcus spp. is able to evade recognition by C-type lectin receptors (CLR), such as Dectin-1 and Dectin-2, that could improve the immune response against cryptococcosis. These CLR triggers an intracellular signaling pathway that promotes the recruitment of Syk kinase to initiate signaling cascades that activate cellular pathways crucial to induce protective antifungal cell immune responses. The central role of CLR in immunity to fungal pathogens is the major research focus of Brown's laboratory, who are an internationally-leading team studying the signaling pathways and cellular responses induced by these receptors. In this application, we wish to combine the expertise and research activities of the Brown and da Silva teams to generate novel GXMR-Dectin-1-CAR and GXMR-Dectin-2-CAR constructs and determine their effectiveness in controlling Cryptococcal spp. infections. The GXMR-Dectin-1-CAR and GXMR-Dectin-2-CAR constructs will be used to modify NK-92 cells. Previous studies demonstrated an enhanced capacity of C-type lectin receptors signaling pathways to induce the movement of perforin in NK cells, including the NK-92 cell line, toward the contact site with the target cells. Thus, the activation of the Dectin-1 and Dectin-2 signaling pathway will enhance polarization of high levels of perforin to the synapse between NK-92 cells and the fungus, improving the directed cytolysis of the fungal pathogen. The investigators have all the required knowledge to develop the new CARs, moreover, the capacity of Dectin-1 and Dectin-2 to recruit Syk will enable GXMR-Dectin-1-CAR and GXMR-Dectin-2-CAR to polarize an augmented production of perforin toward the contact site between modified NK cells and Cryptococcus spp.. Then, perforin-dependent killing of Cryptococcus spp. by these modified NK-92 cells will be evaluated to control the in vitro and in vivo infection with Cryptococcus spp. (AU)