Immune cells that are engineered to attack cancer cells, known as CAR-T cells, are used to treat some types of blood cancer. However, only about 5 percent of hospitals in the United States have the ability to generate and deliver CAR-T cells to patients. For many patients, this means the cells need to be frozen and shipped long-distance.
To help make this type of therapy accessible to more people, researchers at MIT have developed a new way to protect the cells from damage that can occur when they are frozen for storage and shipment. Their technique significantly reduces the use of a chemical preservative that is now used to protect the cells, which should make it easier for more hospitals to provide this treatment option to patients.
Instead of treating the cells with a cryoprotective chemical that has to be removed before treatment, the researchers were able to preserve them using a nontoxic antifreeze sugar.
"With this approach, you could theoretically just thaw the cells and then inject them, without any extra processing steps. We think that could allow a lot more cancer treatment centers to be able to give CAR-T cell therapy," says Ana Jaklenec, a principal investigator in MIT's Koch Institute for Integrative Cancer Research and one of the lead authors of the study, which appears this week in Trends in Biotechnology .
In the study, the researchers showed that cells preserved using this process had higher survival rates and could be successfully used to treat lymphoma and glioblastoma in mice.
Robert Langer, the David H. Koch Institute Professor at MIT, is also a senior author of the paper. MIT postdocs Amy Lee and Khanh Tran are the paper's lead authors.
Preserving cells
To make CAR-T cells, doctors isolate T cells from patient blood samples. These cells are then engineered to express a protein called chimeric antigen receptor (CAR), which can be designed to target specific proteins found on cancer cells.
Then, the cells spend several weeks proliferating until there are enough to transfuse back into the patient. A small number of hospitals are equipped to generate and administer these cells, but most CAR-T cells are generated at centralized lab facilities. Once ready, these cells are frozen and shipped to a hospital or cancer treatment center.
To protect the cells from ice crystals that can damage their membranes, the cells are treated with a chemical called dimethyl sulfoxide (DMSO), which prevents ice crystal formation. This compound must be removed before the cells are transfused, but most hospitals don't have the expertise to do this, which limits their ability to provide CAR-T cell treatment.
The process of removing DMSO can also harm cells, reducing the number of CAR-T cells that are viable and effective. In the new study, the MIT team wanted to find a way to reduce or eliminate DMSO from the process, which could make it easier for these cells to reach more patients.
"We looked at this cell-manufacturing process to see if there are ways to improve it, to increase the efficacy and hopefully eventually get to the point where these cells can be easily distributed to treatment centers," Jaklenec says. "Our goal was to eliminate adding this chemical and really focus on safe excipients like sugars."
The researchers employed two sugars that scientists have previously used to help cells survive cold temperatures. These sugars - trehalose and sucrose - help cells to naturally combat cold by protecting proteins from denaturation and preventing the formation of ice crystals. This antifreeze mechanism is found in many Arctic organisms, such as North American wood frogs, and helps them to survive extreme subzero temperatures.
To get sugar molecules into the cells, the researchers used a technique called electroporation. By applying a small electrical current to the cells, they can briefly create holes in the cell membrane, allowing large molecules such as sugars to pass through. They found that they still needed to add a small amount of DMSO, but not enough that it had to be removed later.
"We believe that our cryopreservation strategy can truly improve the cell therapeutic accessibility because with our strategy, you don't need to remove the cryoprotectants. You could use the cells upon thawing," Lee says.
More effective therapy
The researchers tested this technique on CAR-T cells as well as mesenchymal stem cells, which can differentiate into many other cell types and hold potential for use in regenerative medicine. For both types of cells, a higher percentage of the cells survived the freezing and thawing process when sugars were used as the main cryoprotectant instead of DMSO.
They also used thawed CAR-T cells to treat non-Hodgkin's lymphoma and glioblastoma, in mouse models. Mice treated with CAR-T cells preserved using the new strategy had higher survival rates than mice treated with cells preserved using the conventional DMSO approach.
"Preservation methods for living biotherapeutics have seen limited innovation, remain poorly characterized at scale, and often compromise cell viability and function after thawing," Tran says. "We believe that our findings underscore the importance of thorough characterization and optimization of every stage of cell therapy manufacturing, which could have dramatic impacts on treatment efficacy."
The researchers now hope to work with hospitals to explore whether their new technique could be easily integrated into the process of producing and thawing CAR-T cells.
"If that's successful from a cell viability and functionality standpoint, perhaps we will do a small trial with patients," Jaklenec says.
Vijay G. Sankaran, a professor of pediatrics at Boston Children's Hospital and Harvard Medical School and a Howard Hughes Medical Institute Investigator, who was not involved in the study, says he is excited by the potential applications of the research.
"As a pediatric hematologist and oncologist, many of the cell therapies we use, including CAR-T cells and blood stem cells, require us to collect and freeze a substantial number of cells, so that enough healthy cells are available after thawing for when patients need treatment. This work suggests an innovative approach that could help more cells survive the freezing and thawing process, potentially making these powerful therapies more reliable and effective. Of course, further work will be needed to validate these results in settings where this approach can be clinically applied," Sankaran says.
This work was supported by postdoctoral fellowships from the Ludwig Center at MIT's Koch Institute and the Convergence Scholars Program at the MIT Marble Center for Cancer Nanomedicine.
