Interleukin-6-knockdown of chimeric antigen receptor-modified T cells significantly reduces IL-6 release from monocytes

Abstract Background T cells expressing a chimeric antigen receptor (CAR) engineered to target CD19 can treat leukemia effectively but also increase the risk of complications such as cytokine release syndrome (CRS) and CAR T cell related encephalopathy (CRES) driven by interleukin-6 (IL-6). Here, we investigated whether IL-6 knockdown in CART-19 cells can reduce IL-6 secretion from monocytes, which may reduce the risk of adverse events. Methods Supernatants from cocultures of regular CART-19 cells and B lymphoma cells were added to monocytes in vitro, and the IL-6 levels in monocyte supernatants were measured 24 h later. IL-6 expression was knocked down in regular CART-19 cells by adding a short hairpin RNA (shRNA) (termed ssCART-19) expression cassette specific for IL-6 to the conventional CAR vector. Transduction efficiency and cell proliferation were measured by flow cytometry, and cytotoxicity was measured by evaluating the release of lactate dehydrogenase into the medium. Gene expression was assessed by qRT-PCR and RNA sequencing. A xenograft leukemia mouse model was established by injecting NOD/SCID/γc-/- mice with luciferase-expressing B lymphoma cells, and then the animals were treated with regular CART-19 cells or ssCART-19. Tumor growth was assessed by bioluminescence imaging. Results Both recombinant IL-6 and activated regular CART-19 cells expressing IL-6 triggered IL-6 release by monocytes. IL-6 knockdown in ssCART-19 cells dramatically reduced IL-6 release from monocytes without reducing cytotoxic activity. Mice treated with ssCART-19 cells showed lower IL-6 levels in the serum than mice treated with regular CART-19 cells, but tumor growth and survival were similar between the animal groups. Conclusion IL-6 released from activated CAR T cells may be one of the main initiators of the release of IL-6 from monocytes that can drive CRS. IL-6 knockdown in ssCART-19 cells reduces monocyte release of IL-6 both in vitro and in vivo without affecting antitumor efficacy. The IL-6 knockdown strategy may provide a useful and promising way to improve the safety of CAR T cell therapy.