1. Gallagher, Amy

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A stealthy "masked" crusader is wreaking havoc by sneaking through the body to kill tumors, while sparing healthy tissues and reducing treatment side effects. In a new study, researchers at the University of Chicago Pritzker School of Molecular Engineering have engineered a masked version of the molecule-interleukn-12 (IL-12)-that activates when in the locale of the tumor as cancer cells are eradicated.

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Identifying how to engineer the drug to attack only tumor cells while sparing the rest of the body was the impetus leading to the development of a new version of the molecule through the first-of-its-kind research on earlier clinical trials of IL-12 during the early 1990s, which were halted due to studies that showed severe, toxic side effects.


"Our research shows that this masked version of IL-12 is much safer for the body, but it possesses the same anti-tumor efficacy as the original," said Aslan Mansurov, PhD, a postdoctoral research fellow and first author of the new study, who conducted the IL-12 engineering procedure with Jeffrey Hubbell, PhD, the Eugene Bell Professor in Tissue Engineering.


To reinvigorate the possibility of IL-12 with greater potential, researchers designed a masked molecule with a cap covering the section of IL-12 which normally binds immune cells.


"The cap can be removed only by tumor-associated proteases, a set of molecular scissors found in the vicinity of tumors to help them degrade surrounding healthy tissue," said Mansurov. "When the proteases chop off the cap, the IL-12 becomes active and able to spur an immune response against the tumor."


The masked IL-12 is inactive everywhere in the body except at the site of the tumor, where these proteases can cleave off the mask, he added. This approach significantly reduces treatment-associated side effects.


Reengineering for Enhanced Safety

Cytokines work by modulating or training the immune system how to react to threats through the process of activating killer T cells. One cytokine in particular, immunotherapy drug IL-12, has shown potential as a powerful cancer treatment. However, IL-12 also instructs immune cells to produce a large amount of inflammatory molecules that can damage the body and cause severe side effects, including liver damage.


"The biggest concern with IL-12 is its potent and systemic overactivation of immune cells," said Mansurov. "Those studying IL-12 try to mitigate systemic side effects while localizing the therapeutic effect to the tumor."


The study's main objectives are to examine the safety of the molecule in preclinical models and study the model's anti-tumor efficacy.


"Cancer cells proliferate at a much faster rate than healthy cells do," Mansurov said. "In doing so, they overproduce certain enzymes that help them invade the nearby healthy tissue and metastasize to other parts of the body." Conversely, healthy cells produce fewer of these enzymes.


"My colleagues and I took advantage of one of the main differences between healthy and cancerous tissue, which is the production of an excess of certain enzymes in cancers," he noted. In essence, the researchers were able to design a method that created a safer version of IL-12.


Through a series of experiments, the research showed that the masked molecule did not cause the inflammation attributed to the unmodified IL-12.


"When we tested the effect of the engineered IL-12 in colon cancer, we found that the drug led to the complete elimination of the cancer cells," said Mansurov. "In models of breast cancer studied in the lab, masked IL-12 was even more effective than anti-PD-1 antibody, an immune therapy commonly used in humans."


To further explore the potential utility of the new drug in treating humans, Mansurov and his colleagues turned to melanoma and breast cancer biopsies collected and donated from patients to ensure that human cancers contained high enough levels of tumor-associated proteases to unmask the IL-12. "When the engineered IL-12 was exposed to the biopsy samples, its molecular mask came off, unleashing its full immune power," Mansurov said.


Matrix metalloproteinases (MMPs) are overexpressed in the tumor microenvironment. They degrade the extracellular space, allowing the cancer cells to migrate and metastasize to distant organs. "With this in mind, we masked IL-12 with a cap that covers the part of the molecule that normally binds to immune cells to activate them," he said. "The cap is removed only when it encounters enzymes in the vicinity of tumors. When these enzymes chop off the cap, IL-12 is reactivated and spurs nearby killer T cells to attack the tumor."


The molecular mask is derived from a subunit of the IL-12 receptor (Beta 1) that is fused to IL-12 via a linker that senses proteases such as MMPs. "Once the masked IL-12 molecule comes into contact with MMPs, the linker connecting the mask to IL-12 gets cleaved off, exposing active IL-12 to the nearby T cells," said Mansurov. "When we applied these masked IL-12 molecules to both healthy and tumor tissue donated by melanoma and breast cancer patients, our results confirmed that only the tumor samples were able to remove the cap." This indicated that masked IL-12 could potentially drive a strong immune response against tumors without causing damage to healthy organs.


The researchers examined the safety of the masked IL-12 by measuring liver damage biomarkers in mice. "We found that immune-related side effects typically associated with IL-12 were notably absent in mice treated with masked IL-12 over a period of several weeks, indicating improved safety," said Mansurov.


In breast cancer models, the masked IL-12 resulted in a 90 percent cure rate, while treatment with a checkpoint inhibitor resulted in only a 10 percent cure rate. The most impactful part of the study, however, was the masked IL-12, a biopsied colon cancer mouse model, which showed a 100 percent cure rate.


"In preclinical models, we compared the toxicity and efficacy of the masked molecule to the parental 'naked' molecule," explained Mansurov. "While anticancer efficacies were very equal, toxicity was significantly reduced in the masked IL-12 molecule.


"For decades, the field has hoped that IL-12 could someday become a viable therapeutic in the fight against cancer and we've now shown that it is possible," he stated. "We'd like to translate this molecule to the clinic and are now talking to a number of potential partners to make that happen. [We'd like to have] the opportunity to work with biopsies from patients to show what could happen in a patient so that we can determine if the objective holds true," he said. "While it will take some time to bring this new development to patients, the new treatment is clearly on the horizon."


Amy Gallagher is a contributing writer