IL-1β Induce CAR-T-Associated Coagulopathy Via the Hsp90/Kallikrein/uPA Pathway

Introduction:

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated a significant efficacy in the treatment of acute B lymphoblastic leukemia (B-ALL). Noteworthy, 50%-56.6% of patients may suffer from CAR-T-associated coagulopathy (CARAC), characterized by hypofibrinogenemia and bleeding. However, the underlying mechanism has not been elucidated.

Methods:

We prospectively collected peripheral blood samples at the timepoint of before lymphodepletion, D0, D7, D14, D28 and IL-6 peak after CAR-T cell infusion in two cohorts at Wuhan Union Hospital. Cohort 1 (August 2019 to March 2021) included 20 patients with B-ALL preformed with proteomics as a training set. Cohort 2 (December 2021 to December 2022) included 75 patients with ALL and lymphoma as the validation set. The diagnosis of CARAC is based on the consensus of CARAC experts in 2022. In vitro and in vivo CARAC model of B-ALL were established. Coagulopathy was evaluated by the levels of cytokines, endothelial activation and coagulation-fibrinolysis biomarkers.

Results:

In two cohorts, we observed that about half patients developed CARAC after CAR-T therapy, with distinguished hypofibrinogenemia. Bleeding was the main manifestations, including mucosal and gastrointestinal bleeding. There was no statistical difference in baseline characteristics between CARAC group and non-CARAC group in cohort 1. Different expression proteins in CARAC group before and after lymphodepletion, as well as between the two groups, were highly enriched in inflammatory immune, coagulation, complement, fibrinolysis pathways. Compared to the non-CARAC group, the levels of endothelial activation markers vWF and VCAM1, and inflammatory indexes HSP90AA1 and HSP90AB1 were higher in CARAC group. While the inhibitors of fibrinolysis-kinin system PAI-3, A2M, and zymogen levels of F2, KLKB1, FGA/FGB/FGG, PLG were lower than non-CARAC group. These proteomics results were verified in cohort 2. Considering the close relationship between CRS and CARAC, we treated human umbilical vein endothelial cells with the five most common cytokines of CRS, and found that interleukin (IL)-1β could markedly increase the expression of heat shock protein (Hsp) 90 and urokinase-type plasminogen activator (uPA). Hsp90 inhibitor can inhibit the expression of uPA, and reduce the promoting effect of IL-1β on uPA, while kallikrein can also increase the production of uPA. Furthermore, the CARAC mouse model exhibited significant pulmonary hemorrhage and necrosis, hemorrhagic effusion in the abdominal cavity, and elevated D-Dimer levels in B-ALL mice that received CD19 CAR-T cells. These manifestations can be improved by IL-1β receptor inhibitor (Anakinra), Hsp90 inhibitor (17-DMAG hydrochloride), and kallikrein inhibitor (sebetralstat). Additionally, anakinra could suppress the expression of Hsp90 and kallikrein in vivo, ultimately decreasing the level of fibrinolytic effector uPA and improving coagulopathy.

Conclusion

CARAC is characterized by bleeding and hypofibrinogenemia and is closely related to the inflammation-immune-endothelium-coagulation-fibrinolysis network. IL-1β of CRS promotes the release of Hsp90 by endothelial cells, which subsequently induces hyperfibrinolysis through the kallikrein-uPA pathway.