Clever-1 interference to overcome resistance to immune checkpoint inhibitors

Abstract

Immune checkpoint inhibitors (ICIs) are therapeutic antibodies that target receptors on T cells and their ligands on antigen presenting cells to stimulate the patient’s own immune system. ICI targets include programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1) that normally restrain the activation of acquired immunity. Resistance to ICIs is known to arise from the immunosuppressive tumor microenvironment, which is maintained by a macrophage subset that expresses common lymphatic endothelial and vascular endothelial receptor 1 (Clever-1).

Extracellular vesicles (EVs) are shed by all cells and they originate from the endosomal pathway or budding of the plasma membrane. Due to this, they contain proteins along with other biomolecules that are expressed or endocytosed by the cell of origin. Thus, EVs derived from Clever-1+ macrophages may influence the efficacy of ICIs as Clever-1+ macrophages do.

The aims of this thesis were to study if EVs derived from Clever-1+ cells suppress the effect of anti-PD-1 and anti-PD-L1 ICI antibodies and whether this suppression could be reversed by therapeutic Clever-1 blockade using the anti- Clever-1 antibody FP-1305 or specific Clever-1 knockdown and knockout using siRNA and CRISPR/Cas9, respectively. The ability of EVs to suppress ICI- mediated T cell activation was studied using a cell-based PD-1/PD-L1 luciferase reporter assay. The EVs were isolated from either KG-1 cell or primary macrophage cultures by fractioning conditioned cell culture mediums with size exclusion chromatography to obtain samples containing only EVs.

EVs derived from Clever-1+ cells suppressed ICI-mediated T cell activation in the PD-1/PD-L1 assay. However, using the therapeutic antibody FP-1305 to block Clever-1 on EVs did not overcome the immunosuppressive effect of EVs. Using siRNA to knock down Clever-1 from KG-1-cell-derived EVs did not show improvement in overcoming the resistance to ICIs but using CRISPR/Cas9 to knock out Clever-1 from primary-macrophage-derived EVs did. Taken together, it appears that Clever-1 blockade cannot overcome the immunosuppression caused by Clever-1+ vesicles, suggesting that FP-1305 cannot block Clever-1 on the EV surface.