Scientists have made great progress in harnessing the body's own immune cells to treat so-called liquid tumors, cancers of the blood and lymphatic system. Yet these powerful cell therapies have been no match for solid tumors, which are tough to access and secrete signals that can stifle immune cells that get too close.
In a new study, Stanford Medicine researchers and collaborators report making inroads on a new type of cell therapy that could more easily infiltrate solid tumors and overcome their defenses. They've discovered a way to supercharge natural killer cells — the most vividly named of our immune cells — into a specialized form that can reside inside tissues and kill tumor 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 co-lead authors of the study 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.
The researchers tested the therapy in mice and found that these special natural killer cells slowed the growth of a variety of solid tumors. When given alongside an antibody treatment that helps the natural killer cells target cancer cells, the therapy curbed tumor growth even more.
Natural killer cells also have the advantage of not eliciting an immune reaction when transferred from one person to another — meaning that, unlike most immunotherapies, which are personalized products created from each patient's own cells, a therapy based on these supercharged natural killer cells could be produced in bulk, frozen and given to any patient in need.
"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."
Contradictory evidence
Natural killer cells were first described in the 1970s and named for their ability to immediately recognize and destroy abnormal cells, such as cancerous ones and those infected by virus. Unlike other white blood cells, which include B cells and T cells, natural killer cells don't require prior exposure to a target, making them valuable rapid responders.
Much of immunology research has focused on circulating immune cells — nomadic populations, including B cells, T cells, natural killer cells and more, that travel through the bloodstream to sites of infection and disease. But some of these nomads eventually settle down inside tissues where they develop roles customized to their new 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, for example, can be found in the skin, mucous membranes, lungs and liver. But there was contradictory evidence of their roles there. Some studies found that tissue-resident natural killer cells were sluggish killers and even immunosuppressive, whereas other studies found them to be highly efficient assassins.
"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.
Immunosuppressive tissue-resident natural killer cells are beneficial in some situations, such as in the uterine lining during early pregnancy, where they help prevent an immune attack on fetal cells and aid the placenta to grow. Fighting cancer requires the other kind.
A Goldilocks recipe
The evidence pointed to two distinct types of tissue-resident natural killer cells, but their identities and how they developed were largely a black box.
In the new study, Sunwoo's team isolated circulating natural killer cells from human blood donors and exposed them to different sets of cues — like recipes of cellular signals.
They knew that a key ingredient was TGF-b, transforming growth factor beta, a multifunctional signaling protein emitted by many different cell types, including tumor cells, and involved in the differentiation of many cell types. But a little goes a long way.
"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."
After comparing recipes, they found that TGF-b was necessary to make natural killer cells tissue-resident, but a steady supply of TGF-b produced sluggish killers. Instead, exposing the natural killer cells to short-lived human epithelial tumor cells, which present a fleeting amount of active TGF-b, produced highly efficient killers. Notably, they found that direct contact with the epithelial tumor cells, rather than mere proximity, was required, suggesting other activation signals are also necessary.
"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.
The researchers catalogued the similarities and differences between the two tissue-resident types. Both expressed the surface proteins CD49a and CD103, for example, but only the type that were efficient killers expressed CD39. These cells were also better equipped with the tools used to induce cell death, including perforin, a protein that punches holes in target cells, and granzyme A, a killing agent delivered through these holes.
Controlling tumor growth
With a reliable recipe to yield supercharged natural killer cells, Sunwoo's team showed that these cells could infiltrate tumor organoids grown in a lab dish. When injected into mice, the cells slowed the growth of various solid tumors over days and weeks, including those derived from human melanoma and head and neck squamous cell carcinoma.
The results were most striking when the researchers combined the supercharged natural killer cells with cetuximab, a monoclonal antibody treatment that helps tag certain tumor cells for destruction. Cetuximab is approved for metastatic colorectal cancer and advanced head and neck squamous cell carcinoma, though it doesn't work well as monotherapy, Sunwoo said.
Over a month, a single dose of the combination therapy suppressed tumors in mice much more than either treatment alone and did not appear to have 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."
He and colleagues are now planning a Phase I clinical trial of the combination therapy in patients with advanced squamous cell carcinoma, which could start by the end of the year, pending approval by the Food and Drug Administration.
And he has developed and applied to patent the recipe to transform and multiply the supercharged natural killer cells — technically known as cytotoxic tissue-resident natural killer cells — at scale. Natural killer cells from a single donor could generate about 20 doses of the cell therapy in about 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.
