Too tight! Loosening CAR’s grip on Tregs improves function, researchers find

A new paper from MUSC Hollings Cancer Center researchers is helping to reveal the “rules” for engineering CAR Tregs.

Tregs, or T-regulatory cells, have shown promise in fighting autoimmune diseases like type 1 diabetes, yet that promise has been unrealized for 20 years.

“The hope is that this study can contribute [to fulfilling that promise]. It's possible. We know it's possible. We're going to keep working hard at it,” said Leonardo Ferreira, Ph.D., senior author of the paper published in Molecular Therapy Methods & Clinical Development.

Russell Cochrane, a LOWVELO HCC graduate fellow in the Ferreira Lab, was first author on the paper. He looked at why engineered chimeric antigen receptor (CAR) Tregs, which in theory should function the same as natural Tregs but in an enhanced manner, haven’t been living up to their promise.

“The CAR construct that we currently use - it's not tailored to the Treg biology. Whenever you use a CAR in a Treg, it is not mimicking the natural signaling of Treg biology. There is an alteration to be more prone to inflammatory signaling,” Cochrane said.

Adding to inflammation is the opposite of what the treatment should do.

“I tried to come up with a solution of how to get rid of that unwanted consequence while still having the benefits,” Cochrane said. “What I learned was if I lowered the affinity of the CAR – meaning how tightly it binds to its target – it still suppresses immune responses through multiple mechanisms; however, it lowers the pro-inflammatory consequences from the artificial signaling.”

Being able to harness the Treg's power could mean better treatments for people with autoimmune diseases, in which the immune system is out of control; organ transplants, which require a lifelong regimen of immunosuppressive drugs; and cancer, which thrives in an inflammatory environment.

From CAR-T to CAR Treg

CAR-T cell therapy is currently used successfully for several types of blood cancers. This model uses a patient’s own T-cells, part of the immune system, and adds a CAR to them. The CAR, or chimeric antigen receptor, recognizes and homes in on a specific antigen on the surface of the cancer cells.

Tregs are a subtype of T-cells.

“Even though they're small in number, they're very potent and important in their ability,” Cochrane said. “They do the opposite of what everyone else does, and that's to regulate the immune system. What Leo always likes to say is that they're the generals of the immune system. They make sure no one's going too low and no one's going too high.”

When it comes to cancers that are solid tumors, though, Tregs aren’t so helpful. Tumors co-opt the Tregs into protecting the cancer cells instead of the body.

“I always think solid tumors are the best immunologists that exist because they learn every trick in the book to prevent the immune system from getting to them,” Ferreira said. “So, an understanding of how Tregs work, what makes them happy, is important both to treat autoimmune diseases and organ transplants, but also to better defeat tumors.”

Tregs isolated from blood are polyclonal, meaning they are a mix of cells, each seeing a different target. But to put them to work for specific conditions, they need to be focused. Ferreira pointed to a recent clinical trial conducted at 15 sites across the country that showed that an infusion of polyclonal Tregs didn’t alter the course of type 1 diabetes.

“We know that for Tregs to work they have to be antigen specific. They have to be laser focused. So there's an urgent need in the field to be able to modify and create and manufacture as many antigen-specific Tregs as possible,” he said. “If you put 3 billion polyclonal Tregs into patients, their disease does not change. So we know that it's not a matter of numbers. You can put in as many polyclonal Tregs as you want to, and it will not work.”

Choosing a target

CAR-T cell therapy works by directly targeting cancer cells. CAR-Tregs could take the same tack – assuming there’s a known target. But for autoimmune diseases like type 1 diabetes, researchers don’t know the trigger – meaning they can’t aim for a specific antigen in the same way that CAR-T cell therapy focuses on a single antigen.

When it comes to organ transplants, the goal is to allow the immune system to operate normally – except for a blind spot protecting the transplanted tissue. Instead, in current practice, organ transplant patients must take immunosuppressants to tamp down their entire immune systems so that neither the body nor the transplanted tissue decides to attack the other.

“With conventional CAR-T cells for cancer, the target is a cancer cell – no question there. But for Tregs, because their effect is more multifaceted, you can actually target the cells to be protected,” Ferreira said.

“Or maybe it's better for the field to rethink this. You could target the extracellular matrix so no cells are harmed in the process. You could target the aggressor T-cells directly. Or you could target the antigen presenting cell that orchestrates the immune attack.”

The ultimate goal, Cochrane said, would be a “living therapeutic to locally curb unwanted inflammation without requiring any broad immunosuppressive drugs.”

To get there, though, researchers must understand how to make CAR-Tregs effective. Previous studies have hinted that adjusting the affinity could make a difference, but this is the first study to make a direct comparison between CAR-Tregs that are otherwise identical except for how tightly the CAR binds to its target.

“It's a direct comparison, so it's very solid evidence,” Cochrane said. “And we found that whenever you lower the affinity, it results in lower pro-inflammatory cytokine secretion.”

Ferreira noted that the team also made sure to run tests on both naive Tregs and memory Tregs to be sure that their findings weren’t the result of an odd batch of Tregs but were, in fact, an across-the-board finding.

“There's no subset of Tregs that is somehow resistant to this phenomenon. It’s very consistent,” Ferreira said.

As they continue their work, Ferreira and his team are continuing to investigate how a CAR affects a Treg’s work.

“We're trying to understand the mechanism of the intrinsic signaling [changes] happening,” Cochrane said. “So you have the start result – really tight binding – and the end result, which is pro-inflammatory cytokine secretion. We don't know what happens in the middle.

“We're trying to pinpoint what is occurring in the middle that's causing some of these Tregs to be bad apples. We're performing single-cell analysis – looking at every individual cell at their protein level and their genome level to understand what pathways the Treg is taking so we can better tailor the CAR signaling for Tregs in the future.”

Cochrane, Russell W. et al. High-affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells, Molecular Therapy Methods & Clinical Development, Volume 32, Issue 4, 101385. DOI: 10.1016/j.omtm.2024.101385

This work was supported by Medical University of South Carolina and Hollings Cancer Center startup funds, Human Islet Research Network Emerging Leader in Type 1 Diabetes grant U24DK104162-07, American Cancer Society Institutional Research Grant IRG-19-137-20, Diabetes Research Connection grant IPF 22-1224, and Swim Across America grant 23-1579 to L.M.R.F., and Cellular, Biochemical and Molecular Sciences training grant 5T32GM132055 and Hollings Cancer Center Lowvelo Graduate Fellowship to R.W.C. Supported in part by the Flow Cytometry and Cell Sorting Shared Resource, Hollings Cancer Center, MUSC (P30 CA138313) and the CCND Genomics and Bioinformatics Core, MUSC (P20 GM148302).

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