Manipulation of the FOXO1 Gene Holds Potential to Enhance CAR T-Cell Fitness

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Evan Weber, PhD, an assistant professor of pediatrics at Children's Hospital of Philadelphia, discussed his work on the role of the FOXO1 gene in T-cell persistence and exhaustion.

Evan Weber, PhD, an assistant professor of pediatrics at Children's Hospital of Philadelphia

Evan Weber, PhD
Credit: CHOP

Currently available chimeric antigen receptor T-cell (CAR-T) therapies carry several drawbacks to their efficacy. Among these are the limited persistence of the CAR T-cells and the phenomena of T-cell exhaustion, in which the CAR T-cells lose their potency and functionality over time.

Evan Weber, PhD, an assistant professor of pediatrics at Children's Hospital of Philadelphia, and his colleagues are currently researching the role of the FOXO1 gene in CAR T-cell persistence and exhaustion, with the goal of potentially improving CAR T-cell fitness. Following Weber’s presentation of some of their findings at the American Society of Gene & Cell Therapy (ASGCT) 27th Annual Meeting, held May 7 to 10, 2024, in Baltimore, MD, CGTLive® interviewed him to learn more about their work.

CGTLive: Can you give some background context about your presentation at ASGCT’s 2024 Meeting?

Evan Weber, PhD: The background for my presentation revolves around CAR T-cell fitness. Obviously, CAR T-cell therapy is an emerging modality of cancer immunotherapy that's had remarkable responses in patients with hematologic malignancies. But there are still a lot of challenges that remain, especially for the treatment of solid tumors. One of those challenges is poor T-cell fitness; in other words, the quality of the T-cells that are reengineered to target cancer in these patients. The problem right now is that CAR T-cells become tired or dysfunctional over time and they also fail to persist in a lot of patients. People in the field now think that this loss of persistence and this T-cell dysfunction are major mechanisms that limit CAR T-cells for patients.

My talk tries to address these 2 central pieces of T-cell fitness: persistence and exhaustion. What we have found is that we can hijack T-cell intrinsic programs related to T-cell memory, which is a natural T-cell state, and one that's associated with long persistence and retained functionality—these 2 things that we want for our CAR T-cells. What we have been able to do is reengineer CAR T-cells in such a way where we can promote these T-cell memory-associated transcriptional programs and epigenetic programs to endow our CAR T-cells with the ability to resist exhaustion, to persist long term, and obviously be more efficacious overall.

Can you give an overview of your presentation itself?

The key points from my talk—I think I already mentioned one, which is sort of these core limitations to CAR T-cell therapies revolving around T-cell fitness—the other major message is that memory T-cells, or biology associated with T-cell memory, can be hijacked to promote highly therapeutic T-cells for either CAR T-cell therapies or potentially other types of T-cell-based immunotherapies, like tumor infiltrating lymphocyte (TIL) therapy or even T-cell receptor engineered T-cells. The way that we're able to promote these promemory programs in CART-cells is by overexpressing a transcription factor that we identified called FOXO1. FOXO1 had been implicated in T-cell memory and T-cell persistence in mouse models, but had sort of been understudied in the context of human T-cell biology and especially in settings of cancer immunotherapy. What we found is that we overexpress FOXO1 in human CAR T-cells, we sort of skew them into this more memory-like state: they upregulate surface markers related to T-cell memory and they have transcriptiona land epigenetic features of a memory T-cell. Ultimately, these FOXO1 overexpressing CAR T-cells are able to more effectively clear leukemia or solid tumors in our mouse models. I think overall, they are just more efficacious and potent T-cells.

I think the other major finding from our study is that while FOXO1 overexpression represents this promising approach to broadly enhance T-cell based immunotherapies, we also found that in CAR T-cells or TIL therapy T-cells, endogenous FOXO1 biology is important for patient responses. We performed a correlative analysis looking at endogenous FOXO1 activity in CAR T-cells that either went on to mediate good clinical responses or patient T-cells that went on to mediate poor clinical responses. It turns out that the patients who responded really well to CAR T-cells have a higher FOXO1 activity score in the CAR T-cells before they're infused into the patient, indicating that even endogenous FOXO1 represents this really important biological axis that we can tap into to make CAR T-cells or other types of therapeutic T-cells better for patients.

How would you summarize the main takeaway of this for doctors and the broader healthcare community?

I think the big picture here is that up until this point a lot of the focus in the CAR T-cell field has been on target identification and creating different CAR architectures to promote different types of signaling in T-cells. But I think what our work and many other labs’ work shows now is that the quality of the T-cell is really a strong predictor of how well these therapies actually do in patients. By promoting therapeutic T-cell states, either through transcription factor overexpression or by some other means, we can make these CAR T-cells more efficacious and treat a broader range of patients with different malignancies and different disease severities.

Were there any challenges during this work that you can discuss?

One of the major challenges associated with this study was that this current dogma around transcription factors that promote T-cell memory programs were focused on a different transcription factor called TCF1 (the gene name is TCF7). This is gonna sound familiar to a lot of cancer immunotherapists. TCF7 is associated with good responses to other types of cancer immunotherapy like checkpoint blockade. TCF7 is mechanistically important for memory formation in mouse T-cells and some people think even in human T-cells. Originally, we assumed that enforcing TCF1 activity in human CAR T-cells would provide this incredible memory-like and potent phenotype. But that was not the case. In fact, TCF1 overexpression had almost no impact on the function of CAR T-cells in our study, whereas FOXO1 had this really dramatic and profound impact on T-cell fitness and potency. I think moving forward, especially people working in the human CAR T-cell space need to be cautious about how they're choosing their targets, and really validate their targets in human T-cells, rather than assuming that some of the mouse T-cell work translates one-to-one over to the human T-cell setting.

Is there anything else you would like to share?

One of the major questions that we often receive when we give talks on FOXO1 or other potency-enhancing approaches is whether this is going to be safe for patients. If we translate our FOXO1 overexpression approach are these patients going to experience severe side effects like cytokine release syndrome or neurotoxicity? Or are they more likely to experience those with a more potent T-cell? I think that's a great question. My lab and other labs are sort of keeping this in the back of our minds as we develop these potency-enhancing approaches. Eventually, what I believe we'll see is the combination of potency enhancements with regulatable systems that allow for context-specific enforcement of those potency enhancements or even potency enhancements that occur in a user-defined manner—in a drug-regulatable manner, for example. I think as the field advances, we'll see more potent T-cells, but also T-cells that are safe and more manipulatable in the clinical setting.

This transcript has been edited for clarity.

Click here to view more coverage of the 2024 ASGCT Annual Meeting.

REFERENCES
1. Weber E. Augmenting CAR T cell fitness through memory reprogramming. Presented at: ASGCT Annual Meeting 2024, May 7-10; Baltimore, Maryland.
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