Scientists at Boston University College of Engineering have developed a new form of CAR-T cell immunotherapy that incorporates a built-in safety switch. The CAR-T cells can be switched on and off, or dialed up or down, making it possible to tailor treatment for the patient and prevent overstimulation of the immune system, which can otherwise lead to serious, potential complications.

The new system, called VIPER (Versatile ProtEase Regulatable) CAR-T cells, involves engineering the CAR-T cells so that they can be controlled by giving a patient an antiviral drug that disrupts the cell’s activity, lessening the safety concerns that come with traditional CARs. “We see this as the next generation of this type of therapy,” said Wilson Wong, PhD, a Boston University College of Engineering associate professor of biomedical engineering, who has been studying CAR-T cells for over 10 years.

Wong and colleagues reported on their developments in Cancer Cell, in a paper titled, “High-performance multiplex drug-gated CAR circuits.” The research team included John T. Ngo, PhD, an ENG assistant professor of biomedical engineering, and Ahmad S. Khalil, PhD, an ENG associate professor of biomedical engineering and associate director of the Biological Design Center.

The billions of immune cells that help protect us from diseases can sometimes need a little boost. For decades scientists have been trying to figure out ways to engineer immune cells to better combat aggressive diseases, such as cancer. The development of chimeric antigen receptor (CAR) T cells has the potential to “revolutionize” cancer medicine, the authors suggested. “CAR-T cells are exciting cancer immunotherapy, in which T cells are redirected to tumors by engineering them to express CARs.”

CAR-T cell therapy uses modified immune system T cells. The native cells are removed from the individual’s blood, and the gene for a cancer cell-binding receptor is transferred before the engineered T cells are then replaced back into the patient. The CAR is tailored to match the specific cancer being targeted. When replaced back in the body, these CAR-T cells reenter the bloodstream and replicate, so that they can start to fight the cancer cells. CAR-T cell therapy has been found to be effective for treating certain types of cancer, especially leukemia.

“It is a very exciting technology,” said Wong. But there are problems with safety, he noted, that can make the therapy extremely risky.

At times, CAR-T cells overstimulate the immune system, which can trigger the release of cytokines. This can cause a potentially fatal inflammatory condition known as cytokine release syndrome (CRS). Other serious complications can include neurological difficulties, or other organs in the body being mistakenly targeted by the immune cells. “Current options for controlling such adverse side effects involve systemic immunosuppression through drug administration or eradication of the engineered T cells through activation of a kill switch installed in the T cells,” the authors continued. However, they noted, “While these strategies can mitigate side effects, they adversely interfere with the therapy’s ability to fight cancer, often causing patients to succumb to tumors after the intervention.” And although several drug-inducible CAR technologies have been developed, the authors noted, “ … few are reliant on a clinically approved pharmaceutical agent with a favorable safety profile.”

The approach taken by Wong and colleagues was to create an inducible safety switch that is built into the CAR-T cell design, but which is activated by an already approved drug.

In all CAR-T cells, part of the receptor sticks out of the cell membrane, while part of it is inside the cell. The part sticking outside of the membrane binds with cancer antigens, which then activates the T cell and destroys the cancer cell. The researchers’ newly developed VIPER CAR-T cells are engineered with the NS3 protease, a proteolytic enzyme that is critical to the hepatitis C virus life cycle. They constructed two different systems—one that is switched on at the time the VIPER CARs are transferred back to a patient, and one that is switched off—that work slightly differently, but which can both be turned off or on by the patient taking the FDA-approved drug, grazoprevir (GZV), which is used for treating hepatitis C. When administered, the drug molecule interacts with the inserted protein chain, kicking off a series of reactions in the cell to make it disengage, or activate, depending on which system is being used. “That is the most exciting part of this study, that the antivirals are already FDA approved,” said Huishan Li, PhD, lead author of the paper and a postdoctoral fellow in Wong’s lab as well as the Khalil Lab.

Describing experiments in mouse models to evaluate responsiveness of the ON and OFF VIPER CARs to GZV, and their ability to clear tumors, the authors reported that mice injected with ON VIPER CAR-T cells and treated with GZV fully cleared the tumors within 28 days, while those that did not receive GZV retained a high tumor burden. Conversely, mice receiving OFF VIPER CAR-T cells cleared tumors without GZV, but displayed increased tumor burden when treated with GZV. There was also 0% survival among the mice that didn’t receive GZV with the ON VIPER CAR, and among those that received GZ with the OFF VIPER CAR. In contrast, of the mice receiving the ON VIPER CAR and GZV, or the OFF VIPER CAR without GZV, 100% and 80% of them survived until day 49, respectively.

“These results demonstrate that the ON and OFF VIPER CARs are functional in an in vivo leukemia model,” the team noted. Further tests in mice demonstrated that the OFF VIPER CAR system can, in addition, prevent the onset of CRS.

Scientists have crafted other CAR-T cell systems that are controlled by pharmaceuticals, but this is the first that has two modes of operation—on or off, the team suggests. The two modes would allow doctors to target the cancer more aggressively, and make it possible to dial down the treatment if necessary, Wong said. Alternatively, doctors could be cautious and turn the VIPER CAR-T cells on incrementally. “The use of FDA-approved drugs as inducers in these technologies is beneficial as it can accelerate their clinical use,” the scientists wrote. “Furthermore, the tunability of the ON and OFF VIPER CARs using these drugs allows for the tailoring of T cell activation levels to individual patients’ needs.”

To further test their approach, the research team compared their results to other similar studies, finding that VIPER CAR-T cells outperformed other systems. They also used VIPER alongside other types of CARs within the same T cell, such that the T cell was engineered with two different cancer-fighting receptors. This could potentially allow for the engineered T cells to target two different cancer markers at one time, Wong said, opening the door for even further advancements in cancer gene therapy.

“We not only have a safety control in place, but we also can have multiple versions at the same time,” Wong said. The team’s long-term goal, following further development of the technology, is to bring it to humans in clinical settings.

“We envision that VIPER CAR-T cell immunotherapy will be able to, in real-time, modulate T-cell activity to mitigate complications like CRS and off-target activities,” the investigators concluded. “Our multiplexed, drug-gated CAR circuits represent the next progression in CAR design capable of advanced logic and regulation for enhancing the safety of CAR-T cell therapy. Given the availability of multiple NS3-targeting drugs and the continued development of new inhibitory compounds, this research provides a streamlined framework for the translation and adaptation of antiviral strategies into effective treatment for malignancies.”

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