Runx3-overexpression cooperates with ex vivo AKT inhibition to generate receptor-engineered T cells with better persistence, tumor-residency, and antitumor ability

by Selleckchem | Jun 28 2023

Background: Solid tumors pose unique roadblocks to treatment with chimeric antigen receptor (CAR) T cells, including limited T-cell persistence, inefficient tumor infiltration, and an immunosuppressive tumor microenvironment. To date, attempts to overcome these roadblocks have been unsatisfactory. Herein, we reported a strategy of combining Runx3 (encoding RUNX family transcription factor 3)-overexpression with ex vivo protein kinase B (AKT) inhibition to generate CAR-T cells with both central memory and tissue-resident memory characteristics to overcome these roadblocks.

Methods: We generated second-generation murine CAR-T cells expressing a CAR against human carbonic anhydrase 9 together with Runx3-overexpression and expanded them in the presence of AKTi-1/2, a selective and reversible inhibitor of AKT1/AKT2. We explored the influence of AKT inhibition (AKTi), Runx3-overexpression, and their combination on CAR-T cell phenotypes using flow cytometry, transcriptome profiling, and mass cytometry. The persistence, tumor-infiltration, and antitumor efficacy of CAR-T cells were evaluated in subcutaneous pancreatic ductal adenocarcinoma (PDAC) tumor models.

Results: AKTi generated a CD62L+central memory-like CAR-T cell population with enhanced persistence, but promotable cytotoxic potential. Runx3-overexpression cooperated with AKTi to generate CAR-T cells with both central memory and tissue-resident memory characteristics. Runx3-overexpression enhanced the potential of CD4+CAR T cells and cooperated with AKTi to inhibit the terminal differentiation of CD8+CAR T cells induced by tonic signaling. While AKTi promoted CAR-T cell central memory phenotype with prominently enhanced expansion ability, Runx3-overexpression promoted the CAR-T cell tissue-resident memory phenotype and further enhanced persistence, effector function, and tumor-residency. These novel AKTi-generated Runx3-overexpressing CAR-T cells exhibited robust antitumor activity and responded well to programmed cell death 1 blockade in subcutaneous PDAC tumor models.

Conclusions: Runx3-overexpression cooperated with ex vivo AKTi to generate CAR-T cells with both tissue-resident and central memory characteristics, which equipped CAR-T cells with better persistence, cytotoxic potential, and tumor-residency ability to overcome roadblocks in the treatment of solid tumors.

Comments:

The researchers in this study aimed to address the challenges associated with using chimeric antigen receptor (CAR) T cells for treating solid tumors. These challenges include limited persistence of CAR-T cells, inefficient infiltration into tumors, and an immunosuppressive tumor microenvironment. The researchers proposed a strategy to overcome these roadblocks by combining the overexpression of a transcription factor called Runx3 with the inhibition of a protein kinase called AKT.

The study utilized second-generation murine CAR-T cells that targeted human carbonic anhydrase 9. These CAR-T cells were genetically modified to overexpress the Runx3 gene and were expanded in the presence of AKTi-1/2, an inhibitor of AKT1 and AKT2. The effects of AKT inhibition, Runx3 overexpression, and their combination on CAR-T cell characteristics were investigated using various techniques such as flow cytometry, transcriptome profiling, and mass cytometry. The researchers evaluated the persistence, tumor infiltration, and antitumor efficacy of these modified CAR-T cells in subcutaneous pancreatic ductal adenocarcinoma (PDAC) tumor models.

The results of the study showed that AKT inhibition promoted the generation of CAR-T cells with a central memory-like phenotype, characterized by the expression of CD62L and enhanced persistence. However, these cells had suboptimal cytotoxic potential. On the other hand, Runx3 overexpression in combination with AKT inhibition led to the generation of CAR-T cells with both central memory and tissue-resident memory characteristics. Runx3 overexpression enhanced the potential of CD4+ CAR-T cells and inhibited the terminal differentiation of CD8+ CAR-T cells induced by tonic signaling. AKT inhibition promoted the central memory phenotype, while Runx3 overexpression promoted the tissue-resident memory phenotype. These novel CAR-T cells with AKTi-generated Runx3 overexpression exhibited robust antitumor activity and responded well to programmed cell death 1 (PD-1) blockade in subcutaneous PDAC tumor models.

In conclusion, this study demonstrated that combining Runx3 overexpression with AKT inhibition can generate CAR-T cells with characteristics of both tissue-resident memory and central memory cells. These modified CAR-T cells exhibited improved persistence, cytotoxic potential, and tumor-residency ability, addressing some of the challenges associated with CAR-T cell therapy for solid tumors.