Synthetic promoters to induce immune-effectors into the tumor microenvironment.

Harnessing the immune-system to eradicate cancer is becoming a reality in recent years. Engineered immune cells, such as chimeric antigen receptor (CAR) T cells, are facing the danger of an overt life-threatening immune response due to the ON-target OFF-tumor cytotoxicity and Cytokine Release Syndrome. We therefore developed synthetic promoters for regulation of gene expression under the control of inflammation and Hypoxia-induced signals that are associated with the tumor microenvironment (TME). We termed this methodology as chimeric-antigen-receptor-tumor-induced-vector (CARTIV). For proof of concept, we studied synthetic promoters based on promoter-responsive elements (PREs) of IFNγ, TNFα and hypoxia; triple PRE-based CARTIV promoter manifested a synergistic activity in cell-lines and potent activation in human primary T-cells. CARTIV platform can improve safety of CAR T-cells or other engineered immune-cells, providing TME-focused activity and opening a therapeutic window for many tumor-associated antigens that are also expressed by non-tumor healthy tissues.

N-Glycans Mediate the Ebola Virus-GP1 Shielding of Ligands to Immune Receptors and Immune Evasion.

The Ebola Virus (EBOV) glycoprotein (GP) sterically shields cell-membrane ligands to immune receptors such as human leukocyte antigen class-1 (HLA-I) and MHC class I polypeptide-related sequence A (MICA), thus mediating immunity evasion. It was suggested that the abundant N-glycosylation of the EBOV-GP is involved in this steric shielding. We aimed to characterize (i) the GP N-glycosylation sites contributing to the shielding, and (ii) the effect of mutating these sites on immune subversion by the EBOV-GP. The two highly glycosylated domains of GP are the mucin-like domain (MLD) and the glycan cap domain (GCD) with three and six N-glycosylation sites, respectively. We mutated the N-glycosylation sites either in MLD or in GCD or in both domains. We showed that the glycosylation sites in both the MLD and GCD domains contribute to the steric shielding. This was shown for the steric shielding of either HLA-I or MICA. We then employed the fluorescence resonance energy transfer (FRET) method to measure the effect of N-glycosylation site removal on the distance in the cell membrane between the EBOV-GP and HLA-I (HLA.A*0201 allele). We recorded high FRET values for the interaction of CFP-fused HLA.A*0201 and YFP-fused EBOV-GP, demonstrating the very close distance (<10 nm) between these two proteins on the cell membrane of GP-expressing cells. The co-localization of HLA-I and Ebola GP was unaffected by the disruption of steric shielding, as the removal of N-glycosylation sites on Ebola GP revealed similar FRET values with HLA-I. However, these mutations directed to N-glycosylation sites had restored immune cell function otherwise impaired due to steric shielding over immune cell ligands by WT Ebola GP. Overall, we showed that the GP-mediated steric shielding aimed to impair immune function is facilitated by the N-glycans protruding from its MLD and GCD domains, but these N-glycans are not controlling the close distance between GP and its shielded proteins.