Cell therapies that harness the immune system have transformed treatment for some cancers of the blood and lymphatic system. Solid tumors, however, have remained much more difficult to treat because they are harder for immune cells to enter and can release signals that weaken nearby immune defenses.
Researchers at Stanford Medicine and collaborating institutions have now developed a strategy aimed at overcoming those obstacles. Their approach transforms natural killer cells, a type of immune cell known for rapidly attacking abnormal cells, into a specialized tissue resident form that can move into solid tumors and destroy cancer cells.
“We show that these tissue-resident natural killer cells infiltrate into the solid tumors much better than conventional natural killer cells. It was very reproducible, very striking and very clear,” said John Sunwoo, MD, the Edward C. and Amy H. Sewall Professor in the School of Medicine and senior author of the study published last month in Science Translational Medicine.
The study’s co-lead authors are Nina Horowitz, PhD, a former doctoral student in otolaryngology; Imran Mohammad, PhD, a postdoctoral fellow in the Sunwoo lab; and June Ho Shin, PhD, a senior scientist in the Sunwoo lab.
Natural Killer Cells Show Promise Against Solid Tumors
The team tested the experimental therapy in mice and found that the modified natural killer cells slowed the growth of several kinds of solid tumors. The effect became stronger when the cells were paired with an antibody treatment that helps guide natural killer cells toward cancer cells.
Natural killer cells may also offer an important practical advantage. They do not typically trigger an immune reaction when transferred from one person to another. Most current immune cell therapies must be individually manufactured from a patient’s own cells, but a treatment based on these modified natural killer cells could potentially be produced in large batches, frozen and made available to many patients.
“It would be almost an off-the-shelf drug,” Sunwoo said. “It could make cell therapy much more accessible to a wider variety of patients.”
Why Tissue Resident Immune Cells Matter
Natural killer cells were first identified in the 1970s. Their name comes from their ability to rapidly recognize and destroy abnormal cells, including cancer cells and cells infected by viruses. Unlike other white blood cells, such as B cells and T cells, natural killer cells do not need to encounter a specific target beforehand, allowing them to respond quickly.
Historically, much of immunology has centered on immune cells circulating in the bloodstream, including B cells, T cells, and natural killer cells. These cells travel throughout the body searching for infection and disease. Some, however, eventually settle inside tissues and take on functions tailored to their local environment.
“For a long time, the study of immunology and disease in humans was concentrated on the blood immune cells,” Sunwoo said. “With the advancement of tools and bioinformatics, we are now starting to look more at what’s going on in tissue. For most immune cells, the tissue is where the action is.”
Tissue resident natural killer cells are found in locations including the skin, mucous membranes, lungs and liver. Scientists have struggled to understand exactly what they do because previous studies have produced conflicting results. Some suggested these cells were relatively weak killers and could even suppress immune activity, while others found that they were highly effective at destroying target cells.
“They may adopt different functions based on certain cues in the microenvironment and in the tissue, and differentiate into a certain kind of sub-population,” Sunwoo said.
In some circumstances, immune-suppressing tissue-resident natural killer cells are beneficial. During early pregnancy, for example, these cells in the uterine lining help prevent the immune system from attacking fetal cells and support placental growth. Cancer treatment, however, requires the more aggressive type.
Finding the Right Cellular Recipe
Evidence suggested that there were two distinct forms of tissue-resident natural killer cells, but researchers did not fully understand how they developed or why their behavior was so different.
To investigate, Sunwoo’s team isolated circulating natural killer cells from human blood donors and exposed them to different combinations of cellular signals.
One important ingredient was TGF-b, transforming growth factor beta. This signaling protein is produced by many cell types, including tumor cells, and plays a role in determining how cells develop. The researchers found, however, that the amount and duration of the signal were critical.
“It’s a Goldilocks kind of thing where if you give just enough of a TGF-b signal, then the natural killer cells become tissue resident with strong toxic activity against malignant cells. If you give too much TGF-b, they’re still tissue resident, but they’re inhibited and dysfunctional, and they don’t kill,” Sunwoo said. “You need it to be presented to the natural killer cells in just the right amount and in just the right manner.”
The experiments showed that TGF-b was required to turn natural killer cells into a tissue resident form. But prolonged exposure produced cells that were poor killers.
A different approach worked much better. The researchers briefly exposed natural killer cells to short-lived human epithelial tumor cells that provided a temporary burst of active TGF-b. That produced tissue-resident natural killer cells with strong tumor-killing activity.
Direct physical contact with the epithelial tumor cells was also essential. Simply placing the cells nearby was not enough, suggesting that additional activating signals were involved.
“These two tissue-resident natural killer cell populations look very similar, and they have some of the same requirements, but their function seems to be on opposite ends of the spectrum,” Sunwoo said.
What Makes the Strongest Killer Cells Different
The team then compared the two types of tissue-resident natural killer cells in detail.
Both types displayed the surface proteins CD49a and CD103. Only the highly effective cancer-killing cells, however, expressed CD39.
The stronger cells also contained more of the molecular machinery needed to kill targets. This included perforin, a protein that creates holes in target cells, and granzyme A, a toxic molecule delivered through those openings.
Slowing Tumor Growth in Mice
Once the researchers established a reliable method for producing the more aggressive natural killer cells, they tested how well the cells could enter tumors.
In laboratory experiments, the modified cells successfully infiltrated tumor organoids grown in dishes. When injected into mice, they slowed the growth of several types of solid tumors over periods of days and weeks. These included tumors derived from human melanoma and head and neck squamous cell carcinoma.
The strongest results came when the modified natural killer cells were combined with cetuximab, a monoclonal antibody that helps mark certain cancer cells for immune attack.
Cetuximab is approved to treat metastatic colorectal cancer and advanced head and neck squamous cell carcinoma, although Sunwoo noted that it does not work especially well when used alone.
A single dose of the combination therapy suppressed tumor growth in mice much more effectively over one month than either treatment by itself. The researchers also did not observe apparent adverse effects.
“Even at day 30, when the other mice were sick, the mice that received the combination seemed very healthy,” Sunwoo said, though he cautioned against extrapolating too much from mice to humans, adding, “This was just proof of concept.”
Toward an Off-the-Shelf Cell Therapy
Sunwoo and his colleagues are now preparing a Phase I clinical trial to test the combination therapy in people with advanced squamous cell carcinoma. The trial could begin by the end of the year, pending approval from the Food and Drug Administration.
Sunwoo has also developed and applied to patent a method for producing and expanding large numbers of the modified cells, technically known as cytotoxic tissue-resident natural killer cells.
According to the researchers, natural killer cells collected from a single donor could produce about 20 treatment doses in roughly two weeks.
“They’ll be cryopreserved, so we can make a bunch of doses and give it to different patients,” Sunwoo said. “There would be no delay.”
Researchers from Ohio State University and Washington University School of Medicine contributed to the work.
The study received funding from the National Institutes of Health (grants R35DE030054, K22CA282364 and R25DC020174), the Tai Tsun Wu Research Fund for Natural Killer Cell Immunotherapy and the Stanford Bio-X Fellowship.
