2016 ASH Annual Meeting
SAN DIEGO—Investigators have found that life-threatening cytokine release syndrome (CRS) and its symptoms are due to the release of macrophage activation syndrome (MAS) cytokines, such as IL-6, IL-8, and IL2RA.
MAS cytokines, at least in vitro, are not made by chimeric antigen receptor (CAR) T cells and are not necessary for CAR T-cell efficacy, the team says.
The cytokines are produced by antigen-presenting cells (APCs) in response to CAR-mediated killing of leukemia.
What’s more, they say, is that this is likely to be different for each CAR structure and possibly even tumor type.
“Understanding these mechanisms, as it relates to our treatment, will be critical to understanding how best to take care of patients and maintain efficacy without toxicity,” said David Barrett, MD, PhD, of the University of Pennsylvania in Philadelphia.
Dr Barrett discussed the relationship between IL-6, CRS, and CAR T-cell therapy at the 2016 ASH Annual Meeting (abstract 654).
“Every CAR system is slightly different,” he explained, “and it’s very important to understand that when we’re talking about efficacy and toxicity.”
Dr Barrett focused on CTL019 (also known as CART19), the CD19-directed 4-1BB CD3ζ CAR used at the Children’s Hospital of Philadelphia (CHOP).
In pediatric acute lymphoblastic leukemia (ALL), CTL019 produced a 93% response rate at 1 month and an overall survival rate of 79% at 12 months in 59 patients.
“Some relapses take place,” Dr Barrett noted. “This is not a perfect therapy, although it has been transformative in the care of patients.”
Eighty-eight percent of the patients experienced CRS of any grade, and 2 died from it. CRS causes high fever and myalgias, and severe CRS causes unstable hypotension that can require mechanical ventilation.
Tocilizumab, the IL-6R blocking antibody, was used in 27% of the patients, generally for grade 4 CRS.
CRS with CTL019
Dr Barrett described CRS in the first patient treated with CTL019 at CHOP in April 2012. The CRS was quite severe, with high fevers and unstable hypotension requiring multiple vasopressors and the need for mechanical ventilation.
“[W]e had no idea what was happening,” he said. “We didn’t understand what the source of the illness was.”
The patient did not respond to steroids or to etanercept, which Dr Barrett indicated is known to help in acute respiratory distress in transplant patients.
“And it was only through some incredible clinical acumen of the treating physicians as well as incredible critical care that was delivered by our ICU that kept this patient alive long enough for us to try tocilizumab,” Dr Barrett continued, “which, thankfully, worked by blocking the most severe side effects in this patient and allowed her to survive.”
Dr Barrett described the course of another patient who developed grade 4 CRS that continued to get worse even after he received tocilizumab, siltuximab, and steroids.
The patient required vasoactive drugs, had seizures, required milrinone, and was placed on a ventilator. One year after receiving CAR T-cell therapy, he recovered.
“This is an incredibly terrifying syndrome to take care of when we don’t understand what’s triggering it or how to stop it,” Dr Barrett emphasized.
Studying CRS
IL-6 is clearly a critical cytokine in the toxicity of CAR T-cell therapy, Dr Barrett said, but IFNγ and other cytokines are also important.
He and his colleagues performed a comprehensive cytokine analysis of pediatric patients treated with CTL019—specifically, engineered T cells composed of an anti-CD19 single-chain variable fragment, CD3ζ activation domain, a 4-1BB costimulatory domain, and transduced with a lentivirus grown on CD3/CD28 beads with a little bit of IL-2.
With that specific CAR, Dr Barrett said they observed a MAS pattern—IFNγ, IL-10, IL-6, and IL-8, which are most elevated in grades 4 and 5 CRS.
“[S]o this pattern, and this clinical syndrome [CRS] was what we believe was driving toxicity in this model,” he said.
To figure out why this was happening, the investigators created 4-1BB CAR-mediated CRS in a mouse model.
The team took leukemia cells from the first patient treated and clinical T cells from her CAR product and put them in an NSG mouse model that they had used for preclinical development.
The investigators then measured cytokine production in the serum of animals 3 and 7 days post-treatment with CTL019.
“And nothing happened,” Dr Barrett said. “The mice didn’t get sick, they cleared their leukemia, and when you looked for cytokines, you found IFNγ, IL-2, and GM-CSF, but you did not find IL-6.”
The team had also included etanercept and tocilizumab in this model, but since the mice didn’t make the toxic cytokines, the antibodies didn’t do anything.
“So why did she get so sick but yet her cancer and her CAR T cells did not make these mice sick and not generate these cytokines?” Dr Barrett asked.
The investigators hypothesized that APCs—not the CAR T cells—were responsible for the toxic cytokines secreted.
“[I]t would be the CAR T-cell-mediated killing of leukemia which would induce this cytokine release from the antigen-presenting cell lineages,” Dr Barrett explained.
To test this theory, the investigators co-cultured CTL019 and Nalm-6 leukemia, with or without cells derived from peripheral blood monocytes.
The team found that IL-6 levels were elevated several logs when CAR T cells killed leukemia in the presence of the APCs.
On the other hand, co-culture of only CTL019 and Nalm-6 produced high levels of GM-CSF, IFNγ, IL-2, and IL-10 but no detectable IL-6 or IL-8.
Transwell in vitro experiments separating CTL019 and Nalm-6 from the APCs showed the same pattern.
The investigators thus confirmed that IL-6 is made by APCs in response to CAR-mediated killing of leukemia.
Nanostring profiling
The team then performed nanostring RNA analysis of separated cell populations recovered from that experiment.
They found that IL-6 and IL-8 are produced by APCs but not by CTL019. IL-2 and IFNγ are produced by CTL019 and not by APCs, and GM-CSF was produced from CTL019.
“There was a clear separation in cytokine production in this model,” Dr Barrett said.
The investigators also observed that the CTL019 nanostring profile was unaffected by proximity to the APCs and all the IL-6 they make.
“CART19 T cells did not seem to care, on a transcriptional level, that all this IL-6 was floating around,” Dr Barrett said.
In contrast, the APCs do change, he said, when CAR T cells are killing leukemia nearby.
“There are dozens and dozens of changes,” he said, “including many in chemokines and IL-6 and IL-8.”
The investigators performed multiple in vitro killing assays and found no difference in CAR T-cell killing potential in the presence or absence of the MAS cytokines.
They also performed peripheral blood analysis of patients experiencing CRS of grades 2 to 5. The team observed that clinical CRS may be divided into MAS and not-MAS patterns. In addition, they detected no IL-6 transcript in any of the CAR T cells isolated from these patients.
“I think we’re going to discover that cytokine release syndrome is a clinical entity that has multiple mechanisms,” Dr Barrett said. “And so it’s very important, when we are talking about our models and talking about our results, to be sure that we’re all speaking the same language.”
