CAR-T cells can be thought of as elite snipers in the immune infantry against cancer. A patient’s native T cells are engineered to be armed with advanced molecular artillery that sharpens their precision in finding and destroying cancer cells. This artillery, the engineered T cell’s chimeric antigen receptor (CAR), homes in on a specific biological marker, or antigen, present on the cancer cell. When the antigen is located, the T cell fires on the tumor.

While impressively fatal against blood and lymphatic cancers, CAR-T cells hit barricades in the harsher solid-tumor environment, inhibiting their success. A chief obstacle is tonic signaling, which occurs when the CAR is continuously alert and the cell is trigger-ready, even if its antigen is nowhere nearby. Like a sniper who has traded sleep for coffee, these CAR-T cells become exhausted and dysfunctional, dehydrating their ability to track and kill their targets.

Efforts to neutralize tonic signaling have revolved around the question: what more can we add to the CAR-T cell to prevent chronic activation? However, Jeremy Bjelajac, PhD, a postdoctoral scholar in the lab of Crystal Mackall, MD, had a novel idea: what if we remove something from the cell, instead?

Doing more with less

Mackall is the Stanford Cancer Institute associate director for cancer immunotherapy, the founding director of the Stanford Medicine Center for Cancer Cell Therapy, and director of the Parker Institute for Cancer Immunotherapy at Stanford. She is the senior author, with Bjelajac as the first author, on a recent Cell publication that details a novel CAR-T cell design based on the immune system’s natural ability to self-regulate and prevent overactivation once an infection has cleared. Without these built-in defenses, the immune system may remain activated and attack healthy tissue, potentially causing toxicity or death. 

“It is remarkable to learn how much control and how many levels and elements are hardwired into biological systems to make sure the immune response isn’t too severe,” Mackall said, “and that the response stops once the virus is gone, so your body regains equilibrium.”

A critical strategy for achieving equilibrium involves proteases, enzymes that cleave, or remove, specific proteins on a cell’s surface and release them into extracellular spaces. This process, called protein shedding, returns the cell to its resting state. Because cells can later replenish the excised proteins by making new copies, protein shedding is reversible. 

Inspired by nature’s efficiency, Bjelajac saw a solution to CAR-T’s longstanding tonic signaling problem: integrate a protease into the T cell to remove the CAR, the source of tonic signaling, after it’s been activated. Like native T cells, the engineered cell gets a break from signaling and can later restore the receptor to resume its search for the target antigen.

Mackall was impressed with the idea’s simplicity. She said, “You look at it, and you say, 'Wow, now that's elegant.’ It's a thing of beauty. We felt that way about this because it's so simple. A lot of the synthetic biology that's out there is sophisticated but complex. When things get complex, they're harder to create, and they take up a lot of space. You have these big receptors, multi-components, and the fact that this was so elemental. And it was so natural.” 

Synthetic biology uses genetic engineering to alter cellular function. It has largely focused on modifying cells by regulating protein expression.This method, called transcriptional control, is accomplished by designing regulatory circuits, a set of genetic instructions that control protein production. While effective, these circuits are bulky and complex, leading to slower cellular processes due to several actions that must be completed before the end goal is achieved. 

“We, as cellular engineers, are always trying to train ourselves only to do things at the right time and right duration and to stop something once it becomes toxic or no longer useful,” Mackall explained. “Jeremy [Bjelajac] said, ‘I don't want to use transcription. I want to use proteases because they are quick and very clean. They only cleave the protein they're designed for, and they go away quickly.’”

Developing the AIR platform

To investigate this approach, Bjelajac used ADAM17, a tightly regulated protease that recognizes proteins responsible for triggering a T cell’s immune response. The response stops after ADAM17 sheds its target proteins, and the T cell returns to its resting state. Bjelajac found that ADAM17’s most efficient protein shedding occurred with a 15-amino-acid sequence found on protein CD62L and located where ADAM17 initiates shedding.

Mackall’s team integrated ADAM17 into the engineered T cell, and the 15-amino-acid sequence was embedded into the CAR. Upon activation, ADAM17 locates the amino acid sequence to shed the receptor, stopping signaling and preventing exhaustion. These steps constitute the activation-induced release (AIR) platform.

“We've tried to take CAR-T cells from liquid cancers like myeloma, lymphoma, and leukemia, and apply them to solid tumors in lung cancer, colon cancer, or sarcomas,” Mackall said, “but we've seen more toxicity there, and the benefits haven't been as great. We're in a bit of a bind where we need to make things more potent, but we can't make them more toxic. This is where the field is struggling to come up with better platforms, better approaches. We think that the AIR platform, because it's regulated, will perhaps give us a more effective, but less toxic, platform.”

Silencing tonic signaling

The AIR platform was integrated into CAR-T cells prone to tonic signaling and tested in cellular assays and mouse models of cancer. Both in vitro and in vivo tests found the platform reduced exhaustion, decreased CAR-T cell death, and improved antitumor potency. 

The tests also revealed that the platform mitigated a common cause of tonic signaling that scientists have been trying to address for more than a decade.

Mackall said, “The chimeric antigen receptors are only supposed to give an activation signal when they find their antigen, but often they bind to each other and aggregate to form clumps. When that happens, they give an activation signal even when their antigen isn’t there. This does all kinds of negative things to the cell, including too much activation.”

The strength of the tonic signaling increases with the number of aggregated receptors. Because the AIR platform sheds receptors upon activation, the number of receptors in the crowd decreases, thus eliminating tonic signaling. Mackall said this worked in every tonically signaling CAR her team tested.

A surprising finding

The team also tested the AIR platform with CARs that don’t tonically signal. To their surprise, in vitro and in vivo tests revealed that these cells were healthier, grew more robustly, and achieved better tumor clearance in leukemia and bone cancer models.

“We didn’t expect that,” said Mackall. “We expected it to work for the tonic signal. We didn’t expect it to work for the non-tonic signal, but we saw that both of them worked better. The reason was that even though they don’t tonically signal, they’re prone to cell death when the cell gets activated, and there was less cell death when the signal got turned off quickly.”

Next-generation approaches

Leveraging the AIR platform for applications beyond tonic signaling, Mackall’s team used it as part of a synthetic biology system called a logic gate that programmed CAR-T cells to attack only after detecting two specific antigens. Many potential target proteins also occur on healthy cells, so requiring the presence of multiple antigens increases the likelihood of killing cancer cells while sparing healthy cells. 

To engineer a logic gate using the AIR platform, the team developed a CAR-T cell with two receptors: one that is active and can signal, and another that is inactive, its signaling restrained by a peptide masking the receptor. When the CAR-T cell encounters a cell expressing the active receptor’s target antigen, the AIR platform is activated to remove the peptide mask from the inactive receptor, freeing it to signal. If the second target antigen is detected, then the CAR-T cell destroys the cancer cell. This approach allows precise targeting and, since the reaction is immediate, streamlined annihilation of malignant cells. 

The team also inserted the AIR platform into receptors that control pathways tumors hijack to suppress T cells. The platform improved antitumor function by temporarily shutting down the pathways upon activation, but because the pathways aren’t permanently deleted, the T cell can still maintain normal function at rest.

Of this approach, Mackall said, “Rather than knocking something out completely, you may just want to have it be shed when the cell gets activated. We think you might be able to do things more safely. One of our hypotheses is that we might have less toxicity.”

Because the AIR platform mimics a naturally occurring process, is self-regulating, and is small compared to intricate synthetic circuits, it may be unlikely to elicit an immune response in patients. Mackall said the next steps involve testing the platform with more complex circuits to address other problems, such as antigen heterogeneity, as well as translating the research into the clinic.

She is optimistic that the simplicity and elegance of the AIR platform’s design will improve the benefit and safety of CAR-T cells in solid tumors. 

“We were only imparting to the cell a level of regulation that's present in normal cells, so we felt like rather than creating something entirely new and synthetic, we were just co-opting a level of regulation that Mother Nature is already using.”

“We were only imparting to the cell a level of regulation that's present in normal cells, so we felt like rather than creating something entirely new and synthetic, we were just co-opting a level of regulation that Mother Nature is already using.”