Safe and potent anti-CD19 CAR T-cells with shRNA-IL-6 gene silencing element in patients with refractory or relapsed B-cell acute lymphoblastic leukemia

Abstract

Severe cytokine release syndrome (sCRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) have limited the widespread use of chimeric antigen receptor T (CAR T)-cell therapy. We designed a novel anti-CD19 CAR (ssCART-19) with a small hairpin RNA (shRNA) element to silence the interleukin-6 (IL-6) gene, hypothesizing it could reduce sCRS and ICANS by alleviating monocyte activation and proinflammatory cytokine release. In a post hoc analysis of two clinical trials, we compared ssCART-19 with common CAR T-cells (cCART-19) in relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL). Among 87 patients, 47 received ssCART-19 and 40 received cCART-19. Grade ≥3 CRS occurred in 14.89% (7/47) of the ssCART-19 group versus 37.5% (15/40) in the cCART-19 group (p = 0.036). ICANS occurred in 4.26% (2/47) of the ssCART-19 group (all grade 1) compared to 15% (2/40) of the cCART-19 group. Patients in the ssCART-19 group showed comparable rates of treatment response (calculated with rates of complete remission and incomplete hematological recovery) were 91.49% (43/47) for ssCART-19 and 85% (34/40) for cCART-19 (p = 0.999). With a median follow-up of 21.9 months, cumulative nonrelapse mortality was 10.4% for ssCART-19 and 13.6% for cCART-19 (p = 0.33). Median overall survival was 37.17 months for ssCART-19 and 32.93 months for cCART-19 (p = 0.40). Median progression-free survival was 24.17 months for ssCART-19 and 9.33 months for cCART-19 (p = 0.23). These data support the safety and efficacy of ssCART-19 for r/r B-ALL, suggesting its potential as a promising therapy.

Figure 1

CAR T manufacturing data. (A) Transduction efficiency of ssCART‐19 group and cCART‐19 group (p = 0.313; t‐test). (B) CD4⁺/CD8⁺ T‐cell ratio of ssCART‐19 and cCART‐19 group (p = 0.440; t‐test). (C) Cytotoxicity of ssCART‐19 and cCART‐19 groups against target cells (p = 0.364; t‐test).

Figure 2

AEs, CRS, and ICANS in patients with 28 days after CAR T‐cell infusion. (A) Number of AEs at any grade for ssCART‐19 and cCART‐19 groups (p = 0.204; t test). (B) Number of grade ≥3 AEs in the ssCART‐19 group and cCART‐19 group (p = 0.026; t test). (C) Number of CRS at any grade for ssCART‐19 and cCART‐19 groups (p < 0.001; Mann–Whitney nonparametric test). (D) Percentage of different grades of CRS for ssCART‐19 and cCART‐19 groups (p = 0.036; Fisher's exact test). (E) Grade of ICANS for ssCART‐19 and cCART‐19 groups (p = 0.009; Mann–Whitney nonparametric test). Percentage of different grades of ICANS for ssCART‐19 and cCART‐19 groups (p = 0.049; Fisher's exact test). (F) Percentage of different grades of ICANS for ssCART‐19 groups and cCART‐19 groups (p = 0.0449; Fisher's exact test).

Figure 3

Cytokine levels in the two groups. (A–G) Scatter plots of the peak concentration of interleukin 6 (IL‐6), IL‐2, tumor necrosis factor‐α (TNF‐α), IL‐4, IL‐10, IL‐17A, and IFN‐γ. (H–L) Dynamic changes in the cytokines IL‐6, TNF‐α, IL‐2, IL‐10, and IFN‐γ within 28 days after CAR T‐cell infusion.

Figure 4

Response rates in the two groups (p = 0.999; t test).

Figure 5

Swimmer plot of 87 patients showing treatment responses after CAR T‐cell infusion.

Figure 6

Response and survival. (A) NRM of the two groups of patients. (B, C) Kaplan–Meier curves of progression‐free survival and overall survival.

Figure 7

Expansion of CAR T‐cells. (A) C _max in the ssCART‐19 and cCART‐19 groups (p = 0.960). (B) T _max in the ssCART‐19 and cCART‐19 groups (p = 0.768). (C) Dynamic expansion of CAR T‐cells within 30 days postinfusion. The results showed no difference in the copies of CAR T‐cells in the ssCART‐19 and cCART‐19 groups (p = 0.438).