Expression in macrophages of chimeric antigen receptor (CAR) specific for Cryptococcus spp to subvert its escape mechanisms

Abstract

Fungal infections commit millions of lives each year and the cryptococcosis has important relevance because affects immunocomprised and healt individuals. Cryptococcosis representing one of the main causes of worldwide morbidity and mortality among HIV+ individuals. The advance in the development of new antifungal drugs against human cryptococcosis is outdated when considering the ability of Cryptococcus spp. to acquire resistance to standard treatment. This factor is exacerbated by the efficient mechanisms of evasion of the host immune response developed by Cryptococcus spp., with emphasis on the subversion of the phagocytic and fungicidal activity of innate immune cells such as macrophages. Therefore, the current proposal aims to bioengineer alveolar macrophages via chimeric antigen receptor (CAR) to promote the direct recognition of Cryptococcus spp. and, concomitantly, induce a cellular activation characterized by high fungicidal activity. These two approaches will involve the GXMR-CAR construct, a CAR with a GXM-specific recognition portion present in the capsule of Cryptococcus spp. The GXMR-CAR construct will be modified to contain the hinge/transmembrane portions and signal transduction domain from the Dectin-1 receptor (GXMR-Dectin-1-CAR-M) or from the Fc³R receptor (GXMR-Fc³R -CAR-M). These new GXMR-CAR constructs will have their coding sequences cloned into a lentiviral vector that will allow the production of lentiviral particles to be used in the modification of AMJ2-C11 and MH-S alveolar macrophages. These modified cells will have their phagocytic capacity evaluated in the presence of Cryptococcus spp., as a way of measuring the effects of the new GXMR-CAR constructs in the redirection of macrophages against the fungus. In continuity, the ability of the GXMR-CAR constructs, through the signaling pathway triggered by the dectin-1 and Fc³R receptor, to induce Syk activation will be highly relevant in the phagosomal maturation process and consequent formation of the phagolysosome after engulfment of the fungus. This process is strongly dependent on the role of Syk, and Cryptococcus spp. drastically subverts the phagosomal maturation process. The present proposal also proposes a sophisticated way of associating Syk activation, via the GXMR-CAR constructs, and the up-regulation of the Syk molecule in a way conditioned by the recognition of Cryptococcus spp. This regulated expression of Syk will occur via a fifth-generation CAR, called SynNotch receptor, which will contain the GXM-binding domain of Cryptococcus spp. Therefore, the current proposal aims to bioengineer alveolar macrophages with different CARs that promote the recognition of Cryptococcus spp. and act on the expression and activation of Syk which is a key molecule in triggering phagocytic and fungicidal activity against infection by Cryptococcus spp. The development of this therapeutic strategy presents an innovative character and is of high importance in clinical studies in the context of cryptococcosis.