ARTICLE IN BRIEF
In a small phase 1 trial, investigators found that the infusion of autologous regulatory T-lymphocytes in ALS patients correlated with slower disease progression.
The idea that neuroinflammation could be a key player in amyotrophic lateral sclerosis (ALS) has generated research in animal models of the disease for almost two decades. But now for the first time the hypothesis has been tested in a small phase 1 clinical trial involving ALS patients.
In an effort to boost the body's anti-inflammatory response and knock down inflammation, a team of investigators in Boston and Houston infused expanded regulatory autologous T lymphocytes (Tregs) into three ALS patients. The technique, which was safe and tolerable, seemed to stabilize or slow ALS symptoms, according to the study published online July 1 in Neurology: Neuroimmunology & Neuroinflammation.
THE STUDY RATIONALE
Stanley H. Appel, MD, FAAN, the Edwards distinguished endowed chair for ALS research and chair of the department of neurology and co-director of the Houston Methodist Neurological Institute, and his colleagues decided to study the immune system of ALS patients based on observations that patients and their families shared an unusually high number of immunological diseases, including thyroid disease, multiple sclerosis, myasthenia gravis, aplastic anemia, polymyositis, Sjögren syndrome, systemic lupus erythematosus, and ulcerative colitis. That started them thinking about the role of inflammation on the disease process, and the animal research began to show that they were heading in the right direction.
In the laboratory, the Texas researchers crossed a mutant superoxide dismutase 1 (SOD1) ALS mouse with a strain that lacks adaptive immune cells. They had thought that by wiping out the proinflammatory T effector cells, the mice would live longer. But on average the ALS mice died a month sooner. That meant that they had knocked out a population of protective cells. To rule out the possible role of B lymphocytes, they crossed an mSOD1 mouse with a CD4 knockout, which wiped out T cells.
“We suspected the effect was due to Tregs, which are CD4 cells, but the mSOD mice were originally crossed with Rag2-/- which remove both CD4 T cells and B cells. Thus, we had to rule out that the effect was not due to B cells, which make antibodies and are not immunosuppressive like Treg cells,” Dr. Appel explained. “The cross of mSOD with CD4-/- brought early death just as the mSOD cross with the RAG2-/- did, thereby ruling out B cells.”
They looked at T-lymphocyte circulation in blood and found some variability but not too far off from what they'd observed in healthy people. They knew that there was also variability in the progression of ALS: Some progressed quickly, and others lived with the disease much longer. It turned out that the people who progressed faster and died sooner were those with significantly depleted Tregs.
Forkhead box P3 (FOXP3), a transcription factor and marker for Tregs, was also low. Further work showed that Tregs were not functioning properly in ALS patients, which meant that suppression of T effector cells was markedly impaired.
STUDY METHODS, FINDINGS
For the current study, Dr. Appel and ALS researchers at Massachusetts General Hospital started with three patients with no family history of ALS: one man with bulbar ALS whose disease was progressing rapidly, a woman who was progressing slowly, and another man who was somewhere in the middle. All three had decreased suppressive function.
Beginning in 2016, each patient was brought in for leukapheresis to filter out his or her white blood cells. The researchers would then begin the process of isolating Tregs. Samples were taken from the batch and expanded for 25 days and purified. The Tregs — a million cells per kilogram — were infused back into the patient: (They took blood samples before and after the procedure to test whether the new cells were present in the sample.) The first round of cells was delivered early in the disease process: The time from the first symptom to the first Treg infusion was 14 months in the first patient, 24 months in the second patient, and 38 months in the third. The patients had four infusions, one every two weeks. During the same eight-week period, patients also received subcutaneous injections of interleukin-2 three times a week. The researchers decided to deliver four more doses over four months during later stages of the disease.
The investigators measured suppression and the function of Tregs ex vivo after every infusion. They also assessed the patients over the course of the study using the Appel ALS score (AALS), the ALS Functional Rating Scale-Revised (ALSRS-R), and measures of maximal inspiratory pressure (MIP). The patients were closely followed to identify side effects and changes in progression rates over time.
The researchers collected, expanded, and infused each of the patients' regulatory T lymphocytes to knock down inflammation and slow the disease process. The Treg suppression function was strongly aligned with slowing progression rates: Treg and FOXP3 numbers increased after each infusion and the suppressive function increased. (FOXP3 is a transcription factor and marker for Tregs and was also low in ALS animals and in patients.)
The infusions slowed or stabilized the progression of symptoms on the AALS score, which is a direct measure of walking time, strength, breathing, speech, and swallowing, during both the early and late disease stages. The patient's respiratory function, measured by MIPs, also stabilized during the infusions. (The ALSFRS-R questionnaire was given every two weeks and suggested a stabilization of the symptoms, but it was subject to a placebo effect because the researchers already knew the questions from the previous visits.)
“At the end of the day, there was a beautiful correlation between suppressive function and clinical state,” Dr. Appel said. “When suppression was stable the clinical state also stabilized or improved.”
“You get increased inflammation when you don't suppress the T-effector cells,” he continued. “This is only three patients, but it is still exciting,” said Dr. Appel. “We hit the target. We dramatically enhanced Treg function, but the benefit didn't last.”
Interestingly, the Treg numbers dropped down within a few weeks in the fastest progressing patient with bulbar ALS, the intermediate patient's Treg numbers lasted a few weeks longer, and the woman with the slowest progression maintained the higher Treg numbers for the longest period. After the cells dropped, the patients seemed to progress at the pre-infusion rate. One patient progressed even faster. Even four to six months later, the second round of infusions — given monthly for four months — kicked up Treg numbers and function again, and the suppression lasted the length of the treatment.
“It told us what we needed to know,” said Dr. Appel. “It stopped working when the first infusions were over and then began again when we resumed infusions months later.”
The authors suspect that “circulating functional Tregs may slow disease progression by suppressing proinflammatory monocytes/macrophages and responder T lymphocytes, as well as entering the CNS and suppressing activated microglia.”
There were no events that linked back to the autologous cells, but there were adverse events related to the disease itself, and these need further investigation, said Dr. Appel. The investigators reported an increase in muscle twitching, and infections; pharyngitis in one patient, aspiration pneumonia in another, and upper respiratory and gastrointestinal infections in the third.
The researchers have received approval from funders to try the procedure with more patients. This time, Dr. Appel and his colleagues are thinking that they should begin the treatment earlier and administer at least monthly injections for a year to show that the cells are maintained and help stabilize the disease process.
The first author of the paper Jason R. Thonhoff, MD, PhD, a clinician-scientist at Houston Methodist, is doing research to determine a way to freeze the cells so that they could have an off-the-shelf product.
The next study will include a placebo-controlled blinded phase, where a patient might get their own cells or a placebo for six months and then everyone would get their own enhanced cells for the final six months. The investigators, who were funded by ALS Finding a Cure and the ALS Association, plan on testing a dozen patients, six in Texas and six in Boston.
Although the study included only three patients, the data are “provocative,” said Jonathan Glass, MD, professor of neurology and pathology at Emory University. “The patients plateaued after both treatment periods and declined when the treatment stopped. This treatment is based on a scientific hypothesis and supported by data. We have nothing else to offer patients, so why not push forward [for clinical trials]?”
Commenting on the study, Robert H. Brown, Jr., MD, DPhil, chair of the department of neurology at UMass Medical School, said: “This study by Appel and colleagues is another extremely strong data point supporting the view that the rate of motor neuron death in ALS is powerfully influenced by non-neuronal cells (that is, motor neuron death is non-cell autonomous).
“So, while the present effects are transient, the underlying lesson is important,” Dr. Brown continued. “A long-term concern is that repeated administration of cell therapy is at best cumbersome. Can we identify soluble effectors from the Tregs that can themselves become therapies that are more easily administered? I might add that the same considerations apply to transient benefits that are seen from infusions of mesenchymal stem cells.”
“Emerging evidence does suggest that neuroinflammation plays an important role in ALS,” said Robert G. Miller, MD, FAAN, a clinical professor of neurology and neurological sciences at Stanford University Medical School and director of the Forbes Norris ALS Research Center at California Pacific Medical Center. “Conventional anti-inflammatory agents have not been helpful. This innovative approach to immunomodulation, involves targeting proinflammatory macrophages and monocytes and responder T lymphocytes and possibly also microglial activation. The treatment appears to be well tolerated.
“The question of efficacy, in a phase 1 trial like this, is rarely clear due to small numbers and lack of controls,” Dr. Miller continued. “Moreover, there is increasing evidence about the nonlinearity of the disease with periods of apparent halting of progression in up to 25 percent of placebo controls. This surprising finding makes it more challenging to examine disease modifying drugs in ALS. Thus, we urgently need biomarkers of disease progression to more efficiently detect whether a therapeutic is engaging the target. In this study, the increase in Treg suppression seemed to correlate with slowing of disease progression. This is tantalizing and deserves further study to determine whether this will be a meaningful biomarker and a much-needed therapy.”