Therapeutic Potential of TNFα and IL1β Blockade for CRS/ICANS in CAR-T Therapy via Ameliorating Endothelial Activation

Abstract

Severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) strongly hampered the broad clinical applicability of chimeric antigen receptor T cell (CAR-T) therapy. Vascular endothelial activation has been suggested to contribute to the development of CRS and ICANS after CAR-T therapy. However, therapeutic strategies targeting endothelial dysfunction during CAR-T therapy have not been well studied yet. Here, we found that tumor necrosis factor α (TNFα) produced by CAR-T cells upon tumor recognition and interleukin 1β (IL1β) secreted by activated myeloid cells were the main cytokines in inducing endothelial activation. Therefore, we investigated the potential effectiveness of TNFα and IL1β signaling blockade on endothelial activation in CAR-T therapy. The blockade of TNFα and IL1β with adalimumab and anti-IL1β antibody respectively, as well as the application of focal adhesion kinase (FAK) inhibitor, effectively ameliorated endothelial activation induced by CAR-T, tumor cells, and myeloid cells. Moreover, adalimumab and anti-IL1β antibody exerted synergistic effect on the prevention of endothelial activation induced by CAR-T, tumor cells, and myeloid cells. Our results indicate that TNFα and IL1β blockade might have therapeutic potential for the treatment of CAR-T therapy-associated CRS and neurotoxicity.

Keywords: CAR-T immunotherapy; coagulation; cytokine release; inflammatory response; leakage.

Figure 1

Supernatant of CAR-T/Nalm6 co-culture induced endothelial activation. (A) The cytokine release profile in the supernatant of HUVEC incubated with sCAR-T was determined by Luminex. The Y axis represented Target to Effector ratio. (B) The mRNA levels of endothelial activation-associated markers were determined by RT-PCR. GAPDH was taken as the housekeeping gene and data was expressed as fold changes relative to control. n = 3. (C) The protein expression of E-selectin, VCAM1, and ICAM1 in HUVEC induced by sCAR-T incubation was determined by flow cytometry. (D) The protein level of tissue factor (TF) in HUVEC induced by sCAR-T incubation was analyzed by western blot. (E) The concentration of Ang2 secreted by HUVEC was assessed by ELISA. n = 3. (F) The permeability of endothelial monolayer was determined by Evans blue/BSA assay (EBA). n = 3. (G) The protein levels of indicated proteins were determined by western blot. * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001. ns represents not significant. All data were representative of at least three independent experiments.

Figure 2

TNFα was the main mediator released by engaged CAR-T cells in inducing endothelial pro-inflammatory response. (A) The cytokine in the co-cultured supernatant of CAR-T/Nalm6 (sCAR-T) was analyzed by Luminex. (B) HUVEC were stimulated with TNFα (10 μg/ml), IFNγ (50 μg/ml), IL8 (25 μg/ml), and GM-CSF (50 μg/ml) respectively for 4h. The mRNA expression of endothelial activation-related markers was determined by quantitative RT-PCR. GAPDH was taken as the housekeeping gene and data was expressed as fold changes relative to control. (C) The protein expression of adhesion molecules E-selectin, VCAM1, and ICAM1 was determined by flow cytometry. (D) The protein expression of TF in HUVEC was determined by western blot. (E) The venn analysis of HUVEC stimulated with sCAR-T and TNFα. * represents p < 0.05, ** represents p < 0.01, and **** represents p < 0.0001. ns represents not significant. All data were representative of at least three independent experiments.

Figure 3

RNA expression profiling of sCAR-T-activated endothelial cells. (A) Differentially expressed genes (985 up-regulated and 1140 down-regulated) were shown in the Volcano Plot. (B) GO term enrichment analysis of differentially expressed genes. (C) The top ten significantly enriched pathways were obtained by KEGG analysis. (D) TNF signaling-associated genes were shown in the heatmap.

Figure 4

The effects of adalimumab on endothelial activation induced by sCAR-T. HUVEC were incubated with sCAR-T supplemented with different doses of adalimumab (1 µg/ml, 5 µg/ml, and 10 µg/ml) or isotype control for 4h. (A) The mRNA expression of endothelial activation-related markers was determined by quantitative RT-PCR. (B) The protein expression of E-selectin, VCAM1, and ICAM1 was determined by flow cytometry. (C) The concentration of IL6 and IL8 in the supernatant was determined by ELISA. (D) The protein expression of TF in HUVEC was determined by western blot. (E) The concentration of Ang2 secreted by HUVEC was assessed by ELISA. n = 3. (F) Confluent HUVEC cultured in Transwell were incubated with sCAR-T supplemented with or without adalimumab for 12h. The permeability of endothelial monolayer was determined by EBA. (G) The protein levels of indicated proteins were analyzed by western blot. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001. ns represents not significant. All data were representative of at least three independent experiments.

Figure 5

The cytokines secreted by myeloid cells enhanced sCAR-T-induced endothelial activation. (A) Schematic presentation of CAR-T/Nalm6/PBMC or CAR-T/Nalm6/CD14+ cells co-culture in Transwell. Details refer to the information in the Materials and Methods. (B) The cytokine profiles of co-cultured supernatants were determined by multi-analyte flow assay. (C) The mRNA levels of endothelial activation-associated markers were determined by RT-PCR. GAPDH was taken as the housekeeping gene and data was expressed as fold changes relative to control. n = 3. (D) The protein expression of E-selectin, VCAM1, and ICAM1 was determined by flow cytometry. n = 3. (E) The protein expression of TF in HUVEC was determined by western blot and GAPDH was taken as the loading control. (F) The concentration of Ang2 secreted by HUVEC was assessed by ELISA. n = 3. (G) Confluent HUVEC cultured in Transwell were incubated with sCAR-T supplemented with or without adalimumab for 12h. The permeability of endothelial monolayer was determined by EBA. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001. ns represents not significant. All data were representative of at least three independent experiments.

Figure 6

The synergistic effects of TNFα and IL1β blockade on endothelial activation induced by CAR-T/Nalm6/PBMC co-cultured supernatant. HUVEC were incubated with CAR-T/Nalm6/PBMC co-cultured supernatant supplemented with perceptively or both adalimumab (10 µg/ml) and anti-hIL1β (20 µg/ml) or isotype control for 4h. (A) The mRNA levels of endothelial activation-associated markers were determined by quantitative RT-PCR. GAPDH was taken as the housekeeping gene and data was expressed as fold changes relative to control. n = 3. (B) The protein expression of E-selectin, VCAM1, and ICAM1 was determined by flow cytometry. n = 3. (C) The concentration of IL6 and IL8 in the supernatant was determined by ELISA. (D) The protein expression of TF in HUVEC was determined by western blot. (E) The concentration of Ang2 secreted by HUVEC was assessed by ELISA. n = 3. (F) Confluent HUVEC cultured in Transwell were incubated with sCAR-T supplemented with or without adalimumab for 12h. The permeability of endothelial monolayer was determined by EBA. (G) The protein levels of indicated proteins were analyzed by western blot. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001. ns represents not significant. All data were representative of at least three independent experiments.

Figure 7

FAK inhibition effectively abolished endothelial activation induced by CAR-T/Nalm6/PBMC co-cultured supernatant. (A) The protein level of phosphorylated FAK at tyrosine 397 as well as TF was determined by western blot. (B) The mRNA levels of endothelial activation-associated markers were determined by RT-PCR. GAPDH was taken as the housekeeping gene and data was expressed as fold changes relative to control. n = 3. (C) The protein expression of E-selectin, VCAM1, and ICAM1 was determined by flow cytometry. n = 3. (D) The concentration of IL6 and IL8 in the supernatant was determined by ELISA. (E) The concentration of Ang2 secreted by HUVEC was assessed by ELISA. n = 3. (F) Confluent HUVEC cultured in Transwell were incubated with sCAR-T supplemented with or without adalimumab for 12h. The permeability of endothelial monolayer was determined by Evans blue-BSA assay. (G) The expression levels of indicated proteins were determined by western blot. * represents p < 0.05, ** represents p < 0.01, and **** represents p < 0.0001. ns represents not significant. All data were representative of at least three independent experiments.

Figure 8

Schematic description of the role of endothelial activation in CAR-T therapy-induced CRS and neurotoxicity. The recognition of tumor cells by CAR-T cells leads to the activation of CAR-T cells and the production of cytokines. Among the most abundant cytokines secreted by engaged CAR-T cells, TNFα played a major role in inducing endothelial activation. CAR-T cells and tumor cells induced the activation of myeloid cells, leading to the secretion of IL1β which was another important pro-inflammatory cytokine that induces endothelial activation. In this process, FAK, NF-кB, and MAPK were activated. The blockade of TNFα and IL1β with adalimumab and anti-IL1β as well as the inhibition of FAK activity effectively ameliorated endothelial dysfunction induced by CAR-T/tumor cells/myeloid cells in CAR-T therapy.