Researchers from the University of Rhode Island and Yale University have demonstrated a promising new approach to delivering immunotherapy agents directly to tumors. The method involves tethering an immunotherapy agent called a stimulator of interferon genes (STING) agonist to an acid-seeking molecule called a pHLIP® (pH-low insertion peptide). The pHLIP molecules targets the high acidity of cancerous tumors, delivering their immunotherapy cargo directly to cells in the tumor microenvironment. Once delivered, the STING agonists engage the body’s innate immune response to fight the tumor.

Reporting on their development in Frontiers of Oncology, the team showed that just a single dose of the pHLIP-STING agonist combination eradicated even large, advanced colorectal tumors in mice. The treated animals also developed lasting immunity, such that their immune systems could recognize and fight cancer long after the initial tumors were destroyed. While the researchers caution that results in mice don’t always translate to humans, the findings do lay the groundwork for a potential clinical trial testing the safety and effectiveness in cancer patients.

“STING agonists are an important class of immune-modulators, but research has clearly shown that they often don’t work on their own and need to be targeted in some way,” said Yana Reshetnyak, PhD, a physics professor at URI and a senior author of the newly reported research. “What we show here is that using pHLIP to target tumors through their acidity, we can successfully go after a variety of different cell types within the tumor microenvironment and achieve synergistic and quite dramatic therapeutic effects.” The paper by Reshetnyak and colleagues is titled “Eradication of tumors and development of anti-cancer immunity using STINGa targeted by pHLIP,” in which the team concluded, “We have found that targeted delivery of STINGa to tumor stroma by pHLIP, which senses acidity at the surface of metabolically active cells [10], effectively stimulates an antitumor response with a single administration and immune memory is developed.”

Immunotherapy is an emerging approach to fighting cancer. For cancer to survive and spread, tumors need to hide from the immune system. In some cases, they do this by expressing proteins that act as immune cloaking devices—tricking the immune system into thinking tumor cells are normal, native cells. Immunotherapy aims to disable these cloaking devices.

One way of uncloaking tumors is through use of immune checkpoint inhibitors, drugs that have a proven effective in treating a variety of cancers. “In recent years, successful cancer treatments have been introduced that are based on the use of immune checkpoint inhibitors (ICIs),” the authors noted. “The checkpoint blockade approach directly targets the adaptive immune system, acting to release the brakes on anti-tumor immune T-cells.” However, the authors continued, these drugs don’t work on all tumors. “While they work well on immunologically “hot” tumors with lots of inflammation, they are much less effective in “cold,” non-inflamed tumors. “Unresponsive tumors are immunologically non-inflamed, or “cold” tumors, exhibiting low cytokine expression and a lack of T-cell and NK cell infiltration,” the team continued.

STING agonists were developed as a means to turn cold tumors into hot tumors—making them more susceptible to an immune response. They do that by causing cells to release interferon, a type of red-flag protein that alerts the immune system to foreign invaders.

The approach has shown promise in the lab, but administering STING agonist to patients has proven challenging. “STING agonists are promising but flawed as an approach to using the immune system to fight “cold,” uninflamed tumors,” the authors noted. “Despite significant progress in the development of novel STING agonists (STINGa), applications appear to be challenged by the low efficiency and poor selectivity of these agents,” the investigators noted.

The compounds can also affect healthy cells, leading to significant side effects and only modest therapeutic effects. So while the first clinical trials generally resulted in what the team described as “disappointingly modest efficacy,” they suggest that targeted delivery of STINGa might overcome the difficulties by allowing general administration without causing systemic immune-activation, and resulting in improved pharmacokinetics.

The team suggested that STING agonists targeted specifically to tumor cells—not just cancer cells but also dormant immune cells within a tumor—may have significantly increased effectiveness.

Their approach to achieve this harnesses a pHLIP, peptide derived from bacteriorhodopsin, a membrane protein that enables some single-celled organisms to convert light to energy. Research led by co-author Donald Engelman PhD, at Yale, showed that pHLIP has a special affinity for acidic environments. “When pHLIP encounters a cell membrane with a neutral pH, it will sit on the surface briefly and then pull away,” explained Engelman. “But if it’s in an acidic environment, then the peptide folds into a helix, crosses the cell membrane and stays there.”

Reshetnyak joined Engelman’s lab in 2003, and developed the of trying to use this helix to seek out cancer cells. It’s well known that malignant tumor cells tend to be highly acidic. Along with Engelman and fellow URI physicist Oleg Andreev, PhD, Reshetnyak has been working for two decades to develop pHLIP as a cancer-seeking delivery mechanism.

The team has shown that they can tether molecules to the part of the pHLIP peptide that enters the cell membrane. Those cargo molecules could be diagnostic agents that help doctors to see tumors more clearly, toxins that kill cancer cells, or immuno-modulators like the STING agonist. Because pHLIP only enters cells in highly acidic environments, they can target tumor cells while leaving healthy cells alone. There are currently two ongoing clinical trials testing the safety of pHLIP compounds in cancer patients.

For their newly reported work, the investigators evaluated whether pHLIP could be harnessed to target to cancers immunotherapeutic molecules that cause the immune system to attack the tumor. To test whether targeting via pHLIP would increase the effectiveness of STING agonist activity, the researchers gave 20 mice with small colorectal tumors (100 cubic millimeters) a single injection of the pHLIP-STING agonist. Within days, the tumors disappeared entirely in 18 mice. The team also treated 10 mice with larger tumors (400 to 700 cubic millimeters) using a single injection of pHLIP-STING. In seven of those mice the tumors were eradicated. In comparison, tumors remained in all 10 mice that received injections of untargeted STING agonist, although they demonstrated a modest slowing of tumor growth for a short time.

The results also indicated that the treatment stimulated immune memory in the treated mice. When cancer cells were injected in pHLIP-STING-treated mice that had been tumor-free for 60 days, new tumors failed to develop. That suggests that once the immune system is primed to attack tumor cells, it continues to do so without additional treatment. “A single pHLIP-STINGa injection promoted the development of T-cell immune memory,” the authors wrote.

The high rates of tumor eradication are encouraging, the researchers say, but what’s also encouraging is the fact that the pHLIP-STING agonist appears to be targeting multiple types of tumor cells. Tumors contain more than just cancer cells. Many have a stroma, a kind of coating of non-cancerous cells that forms both a physical and chemical barrier that protects the tumor from the human immune system. In studying tumor structure in the hours following pHLIP-STING agonist injection, the researchers found a marked decrease in stromal cells.

“The stroma was essentially destroyed,” Reshetnyak said. “The fact that we’re modulating the behavior of the wide variety of cells in the tumor stroma as well as the cancer cells themselves means that we’re inducing interferon signaling synergistically in multiple types of cells and treating the entire tumor. That’s the advantage of using acidity as our target: We’re able to go after the whole tumor rather than just certain cell types.”

The authors further noted, “As opposed to delivery targeted to specific receptors on the surfaces of particular cells, pHLIP offers targeting of all (or a majority) of metabolically active cells within the tumor microenvironment. Since the tumor microenvironment is complex, such an approach in targeting and delivery leads to a significant synergistic effect. A single injection of pHLIP with immunoactivator (STINGa) induces production of cytokines, obliterates tumor stroma and increases the tumor pH, which results in the eradication of tumors and the development of immune memory.”

There is more work ahead before a pHLIP-STING agonist treatment can be used in humans, the researchers acknowledged, but these preliminary results are promising. And because pHLIP-based treatments are already approved for clinical trials, the team hopes they will be able to move forward quickly.

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