shRNA-Interleukin-6 Modified CD19-Specific Chimeric Antigen Receptor T Cell Significantly Improves the Safety in Acute Lymphoblastic Leukemia
Authors: Kang Liqing , Tang Xiaowen , Xu Nan , Li Minghao , Tan Jingwen , Qi Wei , Yu Zhou , Lou Xiaoyan , Xue Shengli , Li Xin , Zhang Jian , Chen Jia , Wang Zhenkun , Li Caixia , Cheng Cheng Fu , Wu Depei , Yu Lei 
Published: November 2019

[Background] An urgent need exists to enhance the safety in treating hematologic malignancies with CAR-T therapy by reducing the CAR-T-related cytokine release syndrome (CRS) . Interleukin-6 (IL-6) is a central driver of CRS and neurotoxicity; hence, inhibition of the IL-6 of T cells via gene engineering may improve the safety of CAR-T therapy. [Objective] Investigation of the efficacy and safety of IL-6-targeting short hairpin (sh) RNA in the CART-19 (referred to ssCART-19) to determine whether the IL-6 shRNA in T cells can reduce the severe CRS incidence of ssCART-19 treatment. [Methods]We designed a short hairpin RNA sequence which targets the 3'UTR region of the human IL-6 transcript, and the sequence was added to a CAR construct containing the CD19 target single chain variable fragment (scFv), the EF1a promoter, the co-stimulated domain of 4-1BB and the CD3zeta domain. In vitro study, While there is no significant difference in the transduction efficiency, proliferation ability and cytotoxicity efficacy of ssCART-19 comparing to regular CART-19, there was clear inhibition of the IL-6 expression. IL-6 shRNA mediated gene silence of ssCART-19 significantly inhibited IL-6 gene expression at both the mRNA level (P<0.001) and the soluble cytokines level (P≤0.0001). IL-6 expression profile from ssCART-19 showed consistently maintained the lower level over the entire 150 hours of experiment period compared to regular CART-19 cells (P<0.001 ). And add the supernatants from regular CART-19/Raji co-culture and ssCART-19/Raji co-culture system to the primary induced monocytes, respectively, ssCART-19 could significantly reduce the monocytes derived IL-6 expression levels compared to regular CART-19. In vivo study, the preclinical study showed the consistent results that ssCART-19 significantly reduced the mouse serum IL-6 levels compared to regular CART-19, but with similar anti-tumor efficacy. In the clinical trail, 13 patients with the similar tumor burden baseline administrated with ssCART-19 (n=7) or regular CART-19 (n=6) cells with a dose of 5-10x106 CAR-T cells per kilogram over three consecutive days (10%, 30%, 60% split dose). While all patients from both groups achieved complete response and the CAR-T cells exhibit similar expansion ability, patients treated with ssCART-19 had lower CRS grade and significantly lower IL-6 level in the human serum compared to patients treated with regular CART-19 (the peak value of IL-6, P=0.0285, the IL-6 AUC(0-Tmax), P=0.0217). CRS emerged in 6/6 patients in regular CART-19 cohort and 6/7 patients in ssCART-19 cohort, severe CRS with grade 3 or higher was observed in 83.3% of the patients (5/6) treated with regular CART-19 cohort versus only 42.8% of the patients (3/7) treated with ssCART-19 cohorts. Tocilizumab was given to 66.7 % (4/6) of the patients in the regular CART-19 cohort and two patients needed more than one treatment with tocilizumab. In the regular CART-19 group one patient occurred CRES. There was no CAR T-related death.
[Conclusion]Our study demonstrated that inhibition of CAR-T derived IL-6 expression by shRNA interfering technology could significantly reduce the severe CRS incidence without affecting their immune-oncotherapy efficacy in treating r/r B-ALL patients, which may provide a potential technology to improve the safety profile and promote the extended use of the CAR-T therapy without sacrificing efficacy.

Disclosures
No relevant conflicts of interest to declare.