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

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 (T_PEX) 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 T_PEX 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, T_PEX, 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 (T_EFF) 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 T_EFF 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 T_PEX 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.
• T_PEX 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 T_PEX, 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.