Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer treatment — for some patients. Right now, an oncologist can collect T cells from a patient with leukemia or lymphoma, modify them to target cancerous blood cells, and return them to the patient’s body, where they persist and proliferate as a living drug (1). The treatment strategy is highly effective for many people, but for a significant percentage of patients with blood cancers, CAR T cell therapy ultimately fails.
Julia Fröse wants to improve CAR T cell therapy so it works more often for more people.
credit: Julia Fröse
“About 40 percent of patients go on to experience relapse,” said Julia Fröse, a cancer biologist and a postdoctoral researcher in Michael Hemann’s laboratory at the Massachusetts Institute of Technology (MIT).
To help those patients, Fröse wants to know when and why this cancer treatment fails. That prompted her recent Science Advances study, in which she and her colleagues developed a new strategy for visually tracking CAR T cells in real time in a patient’s body (2).
The MIT team was not the first to tackle this issue, but they took a different approach than other groups whose work preceded theirs. For instance, in 2019 a team led by Martin Pomper, a radiologist at the University of Texas Southwestern Medical Center, inserted a reporter gene in the CAR T cells; they then showed that they could use a small radioactive molecule to visualize that protein — and the CAR T cells that expressed it — on a positron emission tomography (PET) scan (3).
While the reporter gene strategy requires an additional engineering step, Pomper didn’t view that as a major issue. “The CAR T is already like a Frankenstein monster. It’s already a highly engineered cell,” he said. Besides, his team chose a biomarker that clinicians currently use PET scans to detect when looking for evidence of prostate cancer.
However, the Food and Drug Administration (FDA) has already approved several CAR T cell therapies without reporter genes, and Fröse and her colleagues saw an opening for a different tracking strategy. They decided to create a probe that would bind directly to a CAR T cell’s artificially added, cancer-recognizing antigen receptor. “We don’t have to modify the existing, already approved therapy,” she said.
The researchers focused on CD19-CAR T cells, which target a biomarker for B cells in patients with leukemia or lymphoma. Fröse worked with the research group of Mohammad Rashidian, a protein engineer at the Dana-Farber Cancer Institute, to design a molecular probe that could bind to the T cell’s CD19 receptor on one end and that featured a readily modifiable molecular sequence on the other. The customizable end allowed the scientists to try out different signaling attachments.
Designing the probe was the team’s biggest challenge because they had to figure out how much of the CD19 antigen to include, how to keep the probe a reasonable size, and how to simplify its production. Rashidian’s group tried different designs, and then Fröse tested them in cultured cells and mice, using fluorescent dyes to determine whether or not the probe stuck to the CAR T cells. “We went just through a lot of iterations until we found a good design of the probe that worked,” said Fröse.
Once they had a good design, Fröse and her colleagues ensured that the probe wouldn’t interfere with the CAR T cell therapy. After all, they’d designed their probe to bind directly to the antigen receptor that tells a T cell it’s encountered something it should kill. “Was it going to maybe nonspecifically activate them? Or was it going to block their ability to kill tumor cells?” said Fröse.
To address those questions, the researchers put CD19-CAR T cells and their newly designed probe into solutions containing cancerous cells that either expressed CD19 or not. The CAR T cells killed cells with CD19 but not cells without it. But a living body is much more complex than a solution of cells, so the scientists next tried injecting the probe into mice undergoing CD19-CAR T cell therapy for leukemia. After verifying that the probe did not lower the mice’s survival rates, the team felt confident that administering their probe would not interfere with CAR T cell therapy.
So, they tried using it for its intended purpose: to visualize CAR T cells on PET scans. Fröse and her colleagues administered CD19-CAR T cells to mice with leukemia, waited two days, and then injected the animals with a version of the probe that featured radioactive zirconium. A day later, the scientists scanned the mice to see where the T cells ended up.
We’re hoping we can use this for diagnostics in the future to make sure we can see which patients will actually respond to the treatment.
- Julia Fröse, Massachusetts Institute of Technology
Not only did they see strong signals in the spleen and bone marrow, as they expected, but they also quantified differences in signal intensity between mice, which correlated with the animals’ outcomes. Mice whose spleens had relatively dim signals all died within five days of their PET scans, but mice whose spleens had relatively bright signals all lived six days or longer. “We’re hoping we can use this for diagnostics in the future to make sure we can see which patients will actually respond to the treatment,” said Fröse.
Pomper, who was not affiliated with the paper, appreciated the logic of the new study, but he wondered how effective the scientists’ tracking strategy would be in a clinical setting. Although the researchers found that mice cleared about half of the excess probe from their bodies within two hours, the researchers waited a whole day to be confident they were only imaging probes that had bonded to CAR T cells. Pomper noted from his previous experience that clinicians — with limited machine availability and busy schedules — often prioritize more rapid turnarounds. “You have to work things into standard clinical workflow,” said Pomper.
On top of that, Pomper felt that moving to manufacturing and human use might force Fröse and her colleagues to rethink the signaling end of the probe. He noted that the molecular attachment they used to hold zirconium could cause undesired reactions. “There’s also a radiation issue,” said Pomper. Zirconium itself has a relatively long half-life compared to other PET signaling molecules and could expose patients to more radiation than necessary.
But Fröse is aware of the issues with zirconium. “We want to use something that has a shorter half-life,” she said. They plan to do more safety studies as well.
If the team manages to move forward to clinical trials, Fröse is eager to see what else she and her colleagues can accomplish with their new tracking strategy. The probe they designed works specifically for CD19-CAR T cells. “But this kind of probe can definitely be developed for other CAR T cell types, and that’s actually the hope,” said Fröse.
References
- De Marco, R.C., Monzo, H.J., & Ojala, P.M. CAR T cell therapy: a versatile living drug. Int J Mol Sci 24, 6300 (2023).
- Fröse, J. et al. Development of an antigen-based approach to noninvasively image CAR T cells in real time and as a predictive tool. Sci Adv 10, eadn3816 (2024).
- Minn, I. et al. Imaging CAR T cell therapy with PSMA-targeted positron emission tomography. Sci Adv 5, eaaw5096 (2019).
