Abstract

Production and Biological Activity of RBC-derived Extracellular Vesicles Containing STING Agonist (ADU-S100) Using ERYCEV Platform

Karine Senechal, R&D Project Manager, Erytech pharma

RBC-derived extracellular vesicles (RBCEVs) represent a new valuable Drug Delivery System (DDS), due to their intrinsic properties (small size, natural targeting to immune cells, biocompatibility…) and ease of production[1]. This study evaluates the feasibility of producing functional cargo-loaded RBCEVs from ERYCAPS pre-loaded RBCs[2]. To this end, RBCs encapsulating ADU-S100 or empty RBCs processed with ERYCAPS (hypotonic dialysis encapsulation technology) were subjected to physical vesiculation to produce ERYCEV-STINGa and ERYCEV respectively. The RBCEVs produced and purified, analyzed by NTA, revealed a uniform vesicles population of 95 nm mean size and 32 µg/mL mean ADU-S100 concentration in ERYCEV-STINGa. Luminal EVs markers TSG101 and ALIX were successfully detected by western-blot. Additionally, FACS analysis of surface markers showed moderate CD81 and CD235a levels and low CD47 and PS levels. Functionality of produced RBCEVs were successfully demonstrated in vitro. First, uptake of PKH67-labelled ERYCEV was confirmed in both THP-1 monocytes and EMT6 murine tumor cells. Moreover, THP1-derived macrophages phagocytosed pHrodo-labelled ERYCEV-STINGa, which successfully activated STING pathway, demonstrated by luciferase activity in THP1-Dual™ cells and type I interferon (IFN-beta) production. This first proof of concept to produce functional ERYCEV-STINGa supports the development of ERYCEV platform by further investigating pharmacologic properties in vivo and other cargo loading.