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The role of Regnase-1 in CAR T-cell therapy: A potential target to improve responses in ALL

Jun 11, 2021
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Treatment with chimeric antigen receptor (CAR) T cells is an effective treatment for B-cell acute lymphoblastic leukemia (B-ALL). However, many patients relapse due to short-lived survival and function of the CAR T cells.

The master transcription factor TCF-1 is necessary for memory subset formation leading to improved CAR-T–mediated tumor clearance, sustained remissions, and protection against a secondary tumor. TCF-1 is also responsible for the development and maintenance of precursor exhausted T cells (TPEX) and central memory T cells. However, in a tumor microenvironment, it remains poorly understood how the fate of CAR T cells is decided. TCF-1 regulation in CAR T-cell fate determination may identify the targets that support the development of CAR T cells with enhanced persistence and activity. Regnase-1 suppresses TCF-1, consequently limiting the responses of CAR T cells against tumors.

In a study recently published in Blood, Zheng et al. investigated the complex interactions of Regnase-1, TCF-1, and TPEX in the mediation of CAR T-cell persistence and recall responses.1 The key findings are summarized below.

Study design

The study used multiple analyses on several in vivo and in vitro experiments to understand the role of Regnase-1, TPEX, and TCF-1 in CAR T-cell responses.

Methods

  • To assess CAR-T function, human CD19 CAR-transgenic mice were generated. Cell lines included murine luciferease-expressing progenitor B-ALL cells transduced with MSCV-hCD19-IRES-RFP.
  • The murine CAR T-cell analysis included naïve CD4+ or CD8+ T cells from CAR-transgenic mice, transduced with regnase-1, Tcf7, or non-targeting sgRNA vectors to generate non-targeting (WT), Regnase-1–targeting (KO), or double knock-out (DKO) CAR T cells.
  • For human CAR T-cell analyses, CD4+ and CD8+ cells were purified from peripheral blood mononuclear cells of healthy donors, transduced with CD19 CAR lentivirus, and electroplated with Regnase-1–targeting sgRNA (KO CAR) or non-targeting sgRNA (WT CAR). 

Results

Regnase-1 deficiency enhances CAR T-cell persistence, supporting anti-tumor responses:

  • WT and KO CAR cells induced early tumor remission; however, WT CAR-T–treated mice were more disposed to relapse and death.
  • Regnase-1 deficient CAR T cells prevented secondary tumor engraftment in KO CAR-T–treated mice, with survival of up to 40 days.
  • KO CAR T cells also rapidly increased at early time points in tumor-bearing mice and were 100-fold more numerous than WT cells at Day 7, with this ratio preserved up to Day 21.    

Regnase-1 deficiency promotes the formation of memory-like CAR T cells with recall capacity:

  • A higher proportion of effector T cells (TEFF) was seen in mice treated with WT cells, while in those treated with KO CAR-T, memory-like cells were more prominent by Days 14–21.
  • The memory-precursor effector cells were consistently higher in KO CAR T cells, while short-lived effector cells were higher in WT cells.
  • Regnase-1 KO CAR-T cells progressively convert from TEFF phenotype in an early phase into a majorly memory-like population from approximately the seventh day after transfer.

Regnase-1 KO CAR T-cell reprogramming is tumor-antigen dependent:

  • When comparing WT and KO CAR T cells in mice with or without tumor, they found a significantly higher ratio of KO:WT in the presence of a tumor at Day 7. KO cells remained higher in mice with tumor, but interestingly, both types of CAR T cells were exhausted in tumor-free mice.
  • When transferring KO T cells without the CAR construct, T-cell expansion is similar to WT CAR T cells, meaning that the effect seen in KO CAR T cells depends on both Regnase-1 deletion and CAR expression.
  • With transcriptional profiling of KO CAR T cells, they found a tumor-independent upregulation of effector-associated and survival-associated genes and down-regulation of genes associated with T-cell exhaustion and activation-induced cell death in gene upregulation compared to WT.
  • Tumor-dependent changes in KO CAR T cells included the increased expression of effector-associated genes, but at later time points (Day 21), memory-associated gene expression was higher, and effector-associated genes decreased compared to WT. 
  • Thus, the shift in KO CAR T cells from effector to memory-like phenotype is tumor-dependent and can be detected as early as 7 days after transfer.

Targeting Regnase-1 enhances the formation of TCF-1+ CAR T cells:

  • As stated, Reganse-1 suppresses TCF-1; hence, CAR T cells without Regnase-1 of tumor-bearing mice had a higher proportion of TCF-1+ cells with a memory-like phenotype, along with increased TCF-1 expression levels.

Regnase-1 suppresses memory-associated epigenetic programs:

  • Methylation profiles of both WT and KO cells resembled exhausted T cells with few differentiated methylated regions (DMRs) compared to other established T-cell subsets’ epigenetic profiles.
  • Unmethylated profiles were maintained in Regnase-1 KO CAR T cells and were like those found in naïve and central memory T cells, suggesting characterization of memory-associated genes.
  • Effector functions were maintained in both KO and WT cells with DMRs at effector-associated loci. Both WT and KO cells were highly unmethylated and resembled the exhausted T cells profile in DMRs of exhaustion-associated loci.

Improved persistence and TPEX formation in KO CAR T cells is TCF-1–dependent:

  • TCF-1 was expressed in nearly 30% of Regnase-1/TCF-1 DKO CAR T cells, suggesting a bi-allelic disruption.
  • TPEX and memory-associated genes were downregulated in DKO cells, and effector-associated and exhaustion-associated genes were upregulated.
  • In contrast, KO cells were augmented for memory- and naïve-associated gene sets and demonstrated a longer persistence when co-transferred with DKO cells.  

Regnase-1 deletion supports human CAR T-cell expansion and function:

  • Following in vitro stimulation with CD19+ Raji cells, increased expansion was observed in CD8+ and CD4+ Regnase-1–deficient human CAR T cells, even after four rounds of restimulation, supporting the role of Regnase-1 as an essential suppressor of CAR T-cell expansion and tumoricidal activity.
  • A higher proportion of CD25 and CCR7 expression was seen in Regnase-1 KO CAR T cells along with increased proportions of IL-2+, TNFα+, IFNγ+, and Granzyme B+ cells compared to WT CAR T cells.
  • In vivo, reduced tumor burden was seen with human KO CAR T cells in tumor-bearing NSG mice (compared with those treated with WT CAR T cells). At 35 days from the transfer, a higher number of KO CAR T cells were present in the spleen and blood compared to WT cells.
  • Consistently with the murine CAR-T, the human KO CAR T cells also had a higher number of CD8+TCF-1+ and CD4+TCF-1+ cells present.

Conclusion

The findings from this study confirm the vital function of TPEX, Regnase-1, and TCF-1 in facilitating CAR T-cell expansion and persistence. They also identify Regnase-1 as a modulator of human CAR T-cell longevity and potency.

Along with other TCF-1 negative regulators, Regnase-1 may potentially be targeted for improved effectiveness of CAR-T immunotherapies in future clinical practices.

  1. Zheng W, Wei J, Zebley C, et al. Regnase-1 suppresses TCF-1+ precursor exhausted T cell formation to limit CAR T cell responses against ALL. 2021. Online ahead of print. DOI: 10.1182/blood.2020009309

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