Targeted knockdown of Tim3 by short hairpin RNAs improves the function of anti-mesothelin CAR T cells

[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.

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Targeted knockdown of Tim3 by short hairpin RNAs improves the function of anti-mesothelin CAR T cells

10.21203/rs.3.rs-41304/v1 ◽

2020 ◽

Author(s):

Leila Jafarzadeh ◽

Elham Masoumi ◽

Hamid Reza Mirzaei ◽

Khadijeh Alishah ◽

Keyvan Fallah-Mehrjardi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Cytokine Production ◽

Cytotoxic Effects ◽

Car T Cells ◽

Proliferation Capacity ◽

Car T ◽

Before And After ◽

Short Hairpin Rnas

Abstract Background: T-cell immunoglobulin mucin 3 (Tim3) is an immune checkpoint receptor that plays a central role in chimeric antigen receptor (CAR) T cell exhaustion within the tumor microenvironment. This study was aimed to evaluate the effects of targeted-knockdown of Tim3 on the antitumor function of anti-mesothelin (MSLN)-CAR T cells.Methods: To knockdown Tim3 expression, three different shRNA sequences specific to different segments of the human Tim3 gene were designed and co-inserted with an anti-MSLN-CAR transgene into lentiviral vectors. To investigate the efficacy of Tim3 targeting in T cells, expression of Tim3 was assessed before and after antigen stimulation. Afterwards, cytotoxic effects, proliferative response and cytokine production of MSLN-CAR T cells and Tim3-targeted MSLN-CAR T cells were analyzed. Results: Our results showed that activation of T cells and MSLN-CAR T cells led to up-regulation of Tim3. Tim3 knockdown significantly decreased its expression in different groups of MSLN-CAR T cells. Tim3 knockdown significantly improved cytotoxic function, cytokine production and proliferation capacity of MSLN-CAR T cells. Conclusions: Our findings indicate that targeted knockdown of Tim3 allows tumor-infiltrating CAR T cells that would otherwise be inactivated to continue to expand and carry out effector functions, thereby altering the tumor microenvironment from immunosuppressive to immunosupportive via mitigated Tim3 signaling.

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Defining an allogeneic CAR-T approach by shRNA-mediated knockdown of the T-cell receptor.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e14551 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e14551-e14551

Author(s):

David Suhy ◽

Patty Garcia ◽

Vanessa Strings-Ufombah ◽

Natalie Suhy ◽

Peter Roelvink ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

T Cell Activation ◽

Cell Activation ◽

Cell Receptor ◽

Car T ◽

Short Hairpin ◽

Short Hairpin Rnas ◽

Simultaneous Expression

e14551 Background: An allogeneic approach for CAR T-Cell therapy can significantly streamline manufacturing, provide more accessible options to patients as well as enhance safety by reducing the possibility of graft-versus-host-disease. The T-Cell Receptor (TCR) is comprised of multiple subunits and functions to activate T-cells by a signal transduction cascade that is initiated upon antigen binding. Eliminating expression of the endogenous TCR on modified CAR-T-Cells may eliminate the ability to recognize major and minor histocompatibility antigens in the recipient. This study assessed if simultaneous expression of multiple short hairpin RNAs that knockdown levels of individual TCR subunits could result in the loss of TCR expression and TCR-mediated T-Cell activation. Methods: Recombinant DNA producing short hairpin RNAs against the TCR complex was transfected into T-cells. Cell surface TCR was analyzed by FACS. Following CD3 activation or co-culture with B-cells, T-Cell activation was quantified by measuring the levels of IL-2 by ELISA and QPCR. Results: When expressed individually, each shRNA inhibited it’s cognate TCR subunit by up to 93% of the endogenous levels. However, upon simultaneous expression of the shRNAs against the different subunits from the same vector, we observed a near complete depletion of the TCR complex from the cell surface ( > 99%) as measured by FACS analyses. Furthermore, TCR functionality was inhibited when treated cells were stimulated with either CD3 or in B cell co-cultures with Staphylococcal enterotoxins. IL-2 secretion was inhibited to undetectable levels by ELISA by the multi-shRNA treatment and > 98% by qPCR. Conclusions: Though knockdown of any single component of TCR never exceeded 93%, simultaneous knockdown of several TCR subunits abrogated surface TCR and downstream activation suggesting that disruption of stoichiometric expression the subunits prevented TCR formation. The small size of the shRNA expression cassette ( < 2 Kb) permits co-expression from the same lentiviral vector as the CAR. Altogether, these data point to a viable strategy towards generating a single vector approach for the production of allogeneic T-Cells for immunotherapies against certain cancers.

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