Immunotherapies that mobilize a patient’s own immune system to fight cancer have become a treatment pillar. These therapies, including chimeric antigen receptor (CAR) T-cell therapy, have performed well in cancers such as leukemias and lymphomas, but results have been less promising in solid tumors.
A team led by researchers at Penn State College of Medicine has now re-engineered immune cells so that they can penetrate and kill solid tumors grown in the lab. The scientists created a light-activated switch that controls the function of septin-7—a protein associated with cell structure and shape—and incorporated it into natural killer cells, a type of immune cell that fights infections and tumors.
When these cells are exposed to blue light they morph and can then migrate into tumor-spheroids—3D tumors grown in the lab from either mouse or human cell lines—and kill tumor cells. The team suggests that their novel approach could improve cell-based immunotherapies.
“This technology is totally out of the box,” said Nikolay Dokholyan, PhD, the G. Thomas Passananti Professor at the Penn State College of Medicine and professor of biochemistry and molecular biology. “It’s akin to CAR T-cell therapy, but here, the guiding principle is the ability of cells to infiltrate the tumor. I don’t know of another approach that is anything close to this.”
Dokholyan is senior author of the team’s published paper in PNAS, titled “Optogenetically engineered Septin-7 enhances immune cell infiltration of tumor spheroids.” In their paper, the team stated, “Here, we enhance immune cell penetration into tumor spheroids by manipulating septin-7 function in live cells, which might improve cell-based immunotherapies against solid tumors.”
CAR T-cell therapy was first approved by FDA in 2017, and since then has demonstrated encouraging results for some cancers, particularly blood cancers. “The forerunner of cellular immunotherapeutics currently under development is chimeric antigen receptor (CAR) T cell therapies, six of which have been approved by the United States Food and Drug Administration since 2017,” the authors wrote. To create the treatment, T cells are removed from a patient and re-engineered to produce a protein on their surface that binds to a specific target protein on cancer cells. When these CAR T-cells are infused back into the patient, they kill cancer cells with that target protein.
However, CAR T-cell therapy is proving less successful for treating solid tumors, which make up approximately 90% of adult human cancers and 40% of childhood cancers, Dokholyan said. Immune cells can’t infiltrate the dense network of proteins and other cells surrounding the tumor, and the hostile environment inhibits their tumor-fighting abilities. “CAR T cell therapy has shown tremendous success in some liquid tumors, particularly hematopoietic malignancies, whereas it has not performed as well in preclinical studies and clinical trials for solid tumors, with few patients achieving good responses,” the authors further noted. “One of the principal challenges in solid tumor treatment is the physical barrier composed of a dense extracellular matrix, which prevents immune cells from penetrating the tissue to attack intratumoral cancer cells.” In addition, the tremendous diversity among solid tumors makes it difficult to hone in on a specific target protein to attack.
To improve cell-based immunotherapies for solid tumors, Dokholyan said immune cells need to be able to bypass the solid tumor’s defenses. Using computation modeling, the team designed and tested a light-controlled version of septin-7, an internal protein essential for maintaining a cell’s cytoskeleton—the structure that maintains cellular shape and organization. They inserted a light-sensitive domain into septin-7 to create what Dokholyan called “an allosteric regulator.” The authors further explained, “Using protein allosteric design, we reprogram the three-dimensional structure of septin-7 and insert a blue light-responsive light-oxygen-voltage-sensing domain 2 (LOV2), creating a light-controllable septin-7-LOV2 hybrid protein.”
The light-sensitive portion of the protein is located far from the protein’s active site and doesn’t interfere with the structure and function of the protein until it’s triggered. The domain is activated by blue light, which switches the protein function on and off.
The investigators re-engineered human natural killer immune cells with the light-sensitive septin-7 protein. In the presence of blue light, the researchers observed that septin-7’s normal function was disrupted. These re-engineered cells also exhibited a more elongated, spindle-like shape and greater protrusions extending outward, which help the cells interact with their environment and move from one location to another.
“Even though natural killer cells are small, around 10 micrometers, upon activation of this protein with blue light, the immune cells changed shape and can squeeze into tiny holes around three micrometers in size,” Dokholyan said. “That’s enough to infiltrate tumor spheroids and kill them from the inside.”
The researchers tested the re-engineered natural killer immune cells with two types of solid tumor spheroids, one created using human breast cancer cells and the other with human cervical cancer cells. The investigators found that within seven days, the re-engineered immune cells killed the tumor cells. In contrast, control natural killer cells attacked the tumor spheroid from the outside but were unable to breach the tumor, and eventually, the tumor continued to grow. The team in addition re-engineered immune cells from mice and tested them with tumor spheroids made from mouse melanoma cells.
Reporting on their collective studies, the authors stated, “Based on our computational studies and experimental data, we propose that in the dark engineered LCS7 functions as the wild-type septin-7 protein. Upon blue light illumination, the activated LOV2 domain introduces large structural perturbation to the septin-7 structure and impairs septin oligomer formation, which eventually leads to extensive cell protrusions, increased cell polarity, improved cell transmigration efficiency, and enhanced immune cell infiltration into tumor spheroids.”
While the results were robust, Dokholyan emphasized that this work is still in its preliminary stages and more research is needed to evaluate this technology for potential therapeutic use. He said he also hopes to explore other activation cues that could modulate protein function and cellular behavior. The investigators concluded, “Our proposed strategy to enhance immune cell infiltration is compatible with other methodologies and therefore, could be used in combination to further improve cell-based immunotherapies against solid tumors.”
The researchers have filed a provisional application to patent the technology described in the paper.