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CAR-T cells are the god of war among immune cells.
However, God of War has a well-known weakness – it doesn't last.
It is for this reason that more than 50% of patients who respond to CAR-T cell therapy eventually relapse.
And for those patients whose tumors disappeared more than a decade after receiving CAR-T cell therapy, scientists did find evidence of the persistence of CAR-T cells in their bodies.
As a result, making CAR-T cells last longer has become a hot topic in current research.
Today, a team led by Phillip K. Darcy, Paul A. Beavis, and Junyun Lai from the Peter McCollum Cancer Center in Australia[1], and a team led by Evan W. Weber of the University of Pennsylvania and Crystal L. Mackall of Stanford University[2], published back-to-back in the top journal Nature He has published two blockbuster research results and locked the FOXO1 protein as a master switch that regulates the persistence of CAR-T cells.
To put it simply, as long as CAR-T cells overexpress the FOXO1 protein, CAR-T cells will have stem cell-like characteristics and CAR-T cells will have long-lasting anti-tumor activity.
Source: Stanford University School of Medicine
As mentioned above, finding ways to make CAR-T cells last longer is a key issue in the field of CAR-T cell therapy.
At present, there are two main research directions: one is to study the characteristics of CAR-T cells in patients who have achieved durable remission after receiving CAR-T cell therapy [3,4], and then improve CAR-T cell therapy on the basis of these studies, and the other is to directly start with the factors that affect the persistence of T cells to find breakthroughs. Both of the studies presented today take the second approach.
In previous research, Darcy's team found that when culturing CAR-T cells, if IL-15 is added, CAR-T cells can have stronger persistence. This time, they found that IL-15-treated CAR-T cells exhibited a memory T cell phenotype and showed stronger persistence in a mouse model of breast cancer. So, they wanted to know what exactly the effect of IL-15 on CAR-T cells was.
After analyzing the IL-15-treated CAR-T cell multiomics data, it was found that the Foxo1 gene was strongly enriched. Overexpression of FOXO1 protein in CAR-T cells can enable CAR-T cells to have a stem cell phenotype, thereby improving the ability of CAR-T cells to control a variety of solid tumor mouse models. It is important to know that CAR-T cells are often ineffective against solid tumors.
Notably, overexpression of FOXO1 protein preserves the "stemness" and persistence of CAR-T cells to a greater extent than overexpression of TCF1 (a characteristic marker of stem cells), another transcription factor regulated by IL-15.
Screenshot of the homepage of Darcy's team's research paper
Similar to the previous study, Mackall and Weber's team built on previous studies.
A research paper published in Science in 2021 [5] showed that allowing depleted CAR-T cells to rest by transiently inhibiting CAR signaling promotes the formation of a memory-like phenotype and increases chromatin accessibility regulated by the memory transcription factors TCF1 and FOXO1. Therefore, they speculate that TCF1 and FOXO1, two transcription factors, may be the key to mediating the persistence of CAR-T cells.
In fact, TCF1 expression levels have been widely correlated with patient response to CAR-T cells, tumor-infiltrating lymphocytes (TILs), or immune checkpoint inhibitor therapy. FOXO1 directly regulates the expression of TCF1 and other typical immune memory-related proteins, and promotes the formation of memory T cells in mice, and even pharmacological inhibition of AKT, a negative regulator of FOXO1, can cause human CAR-T cells and TILs to produce early memory phenotypes.
The above results suggest that Mackall and Weber's conjecture may be correct. Next, they will need to verify TCF1 and FOXO1, which are required for memory programming and anti-tumor function of human CAR-T cells.
Screenshot of the first page of the Mackall and Weber team's paper
Mackall and Weber's team's first experiment was to overexpress TCF1 and FOXO1, respectively, to see how they affected CAR-T cells. As a result, they found that only FOXO1 overexpression led to increased expression of memory-related cell surface markers and transcription factors.
Notably, under conditions of chronic stimulation, FOXO1 overexpression induces memory-related gene expression programs, while TCF1 overexpression promotes depletion of precursor T cell-like programs. In this way, FOXO1 is what they are looking for.
Sure enough!
After CAR-T cells overexpressed the FOXO1 protein, the ability of CAR-T cells to fight leukemia and solid tumors was enhanced, and the number of CAR-T cells overexpressing FOXO1 increased significantly when the tumor was challenged again. Surprisingly, the "stemness" marker TCF1 was not required, and knocking out the gene encoding TCF1 did not affect the benefits of FOXO1 overexpression to CAR-T cells.
FOXO1 is the key to CAR-T cell anti-cancer
Subsequently, they knocked out the gene encoding FOXO1 in CAR-T cells using CRISPR-Cas9 gene editing technology, and found that CAR-T cells had decreased expansion levels, decreased CD8 positivity rates, decreased memory-related markers, and increased exhaustion-related markers.
In addition, CAR-T cells with knockout of FOXO1-coding genes or CAR-T cells treated with FOXO1 inhibitors showed reduced lethality and/or cytokine secretion when facing tumors, and the ability of CAR-T cells with knockout of FOXO1-coding genes was significantly weakened to control tumors, and the survival rate of tumor mouse models was greatly reduced.
FOXO1 is essential for the function of CAR-T cells
Overall, these two studies suggest that overexpression of transcription factors associated with immune memory can reprogram CAR-T cells to persist and maintain antitumor activity. It is worth mentioning that this is also the first time that scientists have discovered that endogenous FOXO1 protein is required for immune memory-related gene expression and maintenance of optimal anti-tumor function of engineered human T cells.
It is understood that the Darcy team is communicating with clinical experts and is expected to test CAR-T cells overexpressed FOXO1 in clinical practice in the past two years. It is hoped that these two research results can help CAR-T cells expand their territory, especially in solid tumors.
Bibliography:
[1]. Chan, J.D., Scheffler, C.M., Munoz, I. et al. FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy. Nature. 2024. https://doi.org/10.1038/s41586-024-07242-1
[2]. Doan, A.E., Mueller, K.P., Chen, A.Y. et al. FOXO1 is a master regulator of memory programming in CAR T cells. Nature. 2024. https://doi.org/10.1038/s41586-024-07300-8
[3]. Melenhorst JJ, Chen GM, Wang M, et al. Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature. 2022; 602(7897):503-509. doi:10.1038/s41586-021-04390-6
[4]. Anderson, N.D., Birch, J., Accogli, T. et al. Transcriptional signatures associated with persisting CD19 CAR-T cells in children with leukemia. Nat Med, 2023. https://doi.org/10.1038/s41591-023-02415-3
[5]. Weber EW, Parker KR, Sotillo E, et al. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science. 2021; 372(6537):eaba1786. doi:10.1126/science.aba1786
This article was written by BioTalker