Cancer cells are wily, shapeshifting, and ever expanding opponents for the body to fight. But imagine a highly trained assassin aiming for a precise target on the cancer cells: This is the potential that chimeric antigen receptor, or CAR, T cells offer as part of the arsenal of cancer treatments.

There are currently six CAR T cell therapies approved by the Food and Drug Administration, but scientists are still in search of the elusive “off-the-shelf,” or allogeneic, CAR T cell: a single CAR T cell product that can be given to any patient (1). None of the currently approved CAR T cell therapies fit the bill, but researchers at Antion Biosciences think that their gene silencing technology may be the key to a universal CAR T cell therapy.

Antion Biosciences’s technology is up against other heavy hitters for gene editing, such as CRISPR, that have received much more attention in recent years. But the team behind Antion Biosciences is confident that they can keep up in this race to a better CAR T cell.

“Other companies can do it, but just not as efficiently, without question,” said Marco Alessandrini, chief executive officer of Antion Biosciences. “We have something really special.”

The ideal CAR T cell

T cells are just one type of killing machine produced in the body, but they are among the most precise. Typically commissioned to battle infections, T cells are retrained to fight against the body’s own malignant cells for cancer immunotherapy. One way of doing this is by augmenting existing T cells with a CAR designed to recognize a protein found on the cancer cell. For example, to build CAR T cells that can fight B cell lymphomas, researchers design the CAR to recognize CD19, a protein on B cells.

Existing CAR T cell treatments are autologous, meaning that the T cells come from the patient’s own body. Doctors remove the cells from the body, transform them into CAR T cells, and reinfuse them into the patient. This bespoke therapy can be effective but is also costly and resource intensive. “CAR T cells have a lot of downsides, and most of those relate to the difficulty in scaling up,” said Carl DeSelm, an oncologist at Washington University in St. Louis who is not involved with Antion Biosciences. “Having an off-the-shelf approach would make CAR T cells available to far more people.”

With allogeneic T cells, though, a new problem emerges. Like organ transplants, T cells from one person can’t just be arbitrarily infused into another person. They can trigger an immune response where the recipient’s other immune cells attack and destroy the CAR T cells. Unlike most organ transplants, T cells themselves are a type of immune cell, so they can also attack the recipient’s cells.

“You want to avoid that completely,” said Alessandrini. “This is a safety — non-negotiable — thing that has to be done.”

Making sure that a CAR T cell can identify its target, avoid being attacked, and avoid attacking the recipient are necessities, but there are other capabilities that might also be on physicians’ wish lists for their dream CAR T cells: super-strength, longevity, and precision. With multiple tasks to accomplish, the team at Antion Biosciences set out to put together a technology that could do it all. Alessandrini thinks that the problem may be less complex than it seems.

“The gene therapy space is actually very simple,” Alessandrini said. “Gene therapy is quite simply adding a gene or subtracting a gene.”

In the typical process of turning a T cell into a CAR T cell, the first step is to add the gene encoding a CAR. This is required to make autologous CAR T cells too, and current FDA-approved therapies typically accomplish it by delivering the CAR gene as a standalone piece of DNA packaged in a viral vector (2).

You should picture these chromosomes as hot wires. As soon as you cut one, they’re flying all over the place, and they can join up wherever.
- Marco Alessandrini, Antion Biosciences

If the goal is to make an allogeneic CAR T cell, the next step is removing the gene encoding the T cell receptor to keep the CAR T cell from attacking the host’s cells. There’s still a risk of the host’s immune cells attacking the CAR T cell by recognizing a molecule on its surface called the human leukocyte antigen (HLA), which helps the body recognize foreign intruders. This requires an additional step of removing the gene encoding the HLA from the CAR T cell.

Finally, the process of modifying the CAR T cell — especially removing the T cell receptor (TCR) — can weaken the T cell. However, there are additional surface receptors and other molecules that researchers can add to the CAR T cell to make it a more potent cancer killer.

Other companies have devised strategies to make these additions and subtractions with technologies such as CRISPR that make cuts to the cell’s genome to insert or remove the target genes (3). However, Alessandrini noted that cutting the genome comes with increased risk of harmful mutations in patients. “You should picture these chromosomes as hot wires,” he said. “As soon as you cut one, they’re flying all over the place, and they can join up wherever.” Gene editing technologies are also not perfectly efficient. Every addition or subtraction of a gene requires another edit, and each edit has a chance of failure.

That’s why Antion Biosciences has focused on a different approach: combining all the additions and subtractions onto one piece of genetic material that they deliver to the CAR T cells.

From HIV to cancer

The scientists at Antion Biosciences actually first developed their technology years before they began thinking about applying it to cancer. At the time, they were focused on creating stem cells that could be turned into immune cells to treat patients with HIV. The key was being able to remove the gene encoding CCR5, a surface protein that was a known vulnerability against the virus. Researchers at the University of Geneva devised a way to silence the CCR5 gene using microRNA, or miRNA. These short pieces of RNA can bind to the RNA encoding a target protein and tag them for degradation to impede protein production (4).

When the researchers treated mice with these engineered stem cells, they developed resistance to HIV infection (5). But the work halted before ever reaching humans because of lack of investor interest. However, the team at Antion Biosciences realized that there was another potential application with much more interest: CAR T cell development. “From this blossomed a beautiful technology,” said Alessandrini.

Using miRNA to silence genes like TCR and HLA achieved the “subtraction” part of the equation, while existing technology to deliver genes like the CAR would achieve the “addition” steps. Importantly, the sequences of the genes to be added and the miRNA targeting the genes to be silenced could be combined into one piece of genetic material delivered to the T cells via a virus. This avoided the multistep process of CRISPR, as well as the risk of repeatedly cutting the genome.

To accommodate the many different additions and subtractions required for a CAR T cell, Antion Biosciences made the technology more modular, meaning that they could chain many miRNA or gene sequences together to silence many genes. So far, they have successfully modified nine different genes with their multiplexed approach, and they’re testing a version that modifies 10 genes.

While Antion Biosciences has gone after a limited number of candidate genes to improve CAR T cells so far, DeSelm suggested that finding the best targets will be a monumental task in itself. “There's thousands of options that could be argued to be important,” he said. A good place to start might be the combinations of genes targeted in existing CRISPR-based CAR T cells, which DeSelm suggested could be made safer with Antion Biosciences’s multiplexed gene silencing technology.

All I want to do is to see this microRNA in a patient. There's nothing more that I want to see. 
- Marco Alessandrini, Antion Biosciences

There’s another key advantage to this approach: the genes don’t have to be turned all the way off. Using miRNA, they are able to precisely tune how much of the target RNA they degrade so that they still retain some of the protein. This turns out to be essential for HLA: deleting it altogether causes the CAR T cells to catch the attention of natural killer cells. But reducing it by 80 to 90 percent is the sweet spot where the CAR T cells can avoid detection by the immune system, Alessandrini explained.

At the 2023 European Society of Cell and Gene Therapy conference, Antion Biosciences presented data from tests of a CAR T cell designed to target CD19 on B cells that drive certain blood cancers. They engineered the T cells by adding the CAR gene and a safety switch gene designed to selectively kill the cells if needed, while silencing HLA, TCR, and four other genes that could either trigger an immune response or weaken the T cell. These cells killed CD19-expressing cancer cells in a dish and avoided detection by other immune cells. 

The scientists also injected a different version of the CAR T cell into a mouse model, and their preliminary data showed that the silenced genes remained silenced at the desired level even after a month in the mice. “It'll be a question of whether the amount of reduction achieved is enough to make a functional difference,” said DeSelm, noting that more in vivo data will be necessary.

Building a universal cell

Alessandrini hopes that Antion Biosciences’s technology will make a difference even beyond cancer. There are related disease areas, like autoimmunity, where early studies suggest that CAR T cells could be effective, but that’s not the most compelling application to him. He is most excited about developing what he calls a universal donor cell: a stem cell where HLA is silenced so that it can be used to differentiate into any type of cell needed for any patient.

“With a universal construct, we’re going for neurological disease or going for diabetes,” he said. “We’re opening up massive space for us.”

For now, the team’s immediate goal is still in the realm of cancer. In addition to targeting B cell-driven blood cancers, Antion Biosciences is designing CAR T cells that target the CD7 surface marker to treat T cell-driven blood cancers, with the hope of completing animal studies in 2024. Their technology is also attracting the attention of their peers. In 2022, they began a partnership with Allogene Therapeutics, another company working on CAR T cell development. Allogene Therapuetics is using Antion Biosciences’s multiplexed gene silencing technology to design allogeneic CAR T cells that Alessandrini hopes will reach clinical trials in 2025. 

“All I want to do is to see this microRNA in a patient,” Alessandrini said. “There's nothing more that I want to see.”

References

1. National Cancer Institute. CAR T Cells: Engineering Patients’ Immune Cells to Treat Their Cancers. https://www.cancer.gov/about-cancer/treatment/research/car-t-cells (2022).
2. Kalos, M. et al. T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia. Sci Transl Med  3, 95ra73 (2012).
3. Dimitri, A. et al. Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing. Mol Cancer  21, 78 (2022).
4. Myburgh, R. et al. Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction. Mol Ther Nucleic Acids  3, e207 (2014).
5. Myburgh, R. et al. Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice.J Virol  89, 6761-72 (2015).