Researchers at Virginia Polytechnic Institute and State University have developed a new approach to harnessing immunostimulatory cytokine proteins as a potential immunotherapy. Unlike previous methods, the new technique ensures that the cytokines, which are tethered to microparticles, effectively localize within the tumors for weeks, minimizing systemic exposure, while preserving cytokine structure and activity. In multiple, hard-to-treat mouse models of cancer, therapy using the particle-anchored cytokines in combination with immune checkpoint blockade (ICB) immunotherapy resulted in tumor regression, and elicited systemic antitumor immunity against tumor rechallenge. Combination treatment was more effective than either treatment alone.

“When there is a tumor inside the body, the body’s immune cells are being deactivated by the cancer cells,” explained research co-lead Rong Tong, PhD, associate professor in chemical engineering. “The FDA-approved checkpoint blocking antibody helps ‘take off the brakes’ that tumors put on immune cells, while the cytokine molecules ‘step on the gas’ to jump-start the immune system and get an immune cell army to fight cancer cells. These two approaches work together to activate immune cells.”

Tong, together with co-lead Wenjun “Rebecca” Cai, PhD, associate professor in materials science and engineering, and colleagues reported on their development in Science Advances, in a paper titled, “Noncovalently particle-anchored cytokines with prolonged tumor retention safely elicit potent antitumor immunity.” In their report the team concluded, “Collectively, our results indicate that our formulation strategy provides a platform that is likely to be amenable to many potent cytokines and protein therapeutics, which can safely orchestrate adequate immune attacks on solid tumors to improve the clinical management of cancers.”

Last year alone, more than 600,000 people in the United States died from cancer, according to the American Cancer Society statistic cited by the Virginia Tech team. Current cancer treatments, such as chemotherapy, cannot distinguish between healthy cells and cancer cells. When someone with cancer is treated with chemotherapy, the treatment attacks all of the cells in their body, which can lead to side effects such as hair loss and fatigue.

Stimulating the body’s immune system to attack tumors represents a promising alternative approach to treating cancer. And while immune checkpoint blockade (ICB) therapy has been shown to provide long-term remission from numerous cancers in the clinic, the authors noted, “… it is ineffective against many tumors that are poorly infiltrated by immune cells, referred to as immune-excluded or immunologically cold tumors. Combination immunotherapy that can elicit both innate and adaptive immune responses has been actively pursued to treat immunologically cold tumors.”

Cytokines are immunostimulatory agents that could potentially be used to boost immunotherapy in these tumors. Cytokines are small protein molecules act as intercellular biochemical messengers and are released by the body’s immune cells to coordinate their response. “Proinflammatory cytokines, such as IL-2 and IL-12, can stimulate innate and adaptive immune cells and can synergize with other immunotherapies by amplifying and coordinating immune cell responses to overcome immunosuppressive tumor microenvironments (TMEs),” the authors stated. However, harnessing cytokines for anticancer therapy is challenging. While the delivery of cytokines can jump-start immune cells in the tumor, overstimulating healthy cells can cause severe side effects.

“Cytokines are potent and highly effective at stimulating the immune cells to eliminate cancer cells,” Tong added. “The problem is they’re so potent that if they roam freely throughout the body, they’ll activate every immune cell they encounter, which can cause an overactive immune response and potentially fatal side effects.” The authors commented, “Preclinical studies have shown that immunostimulatory cytokines elicit antitumor immune responses but their clinical use is limited by severe immune-related adverse events upon systemic administration.”

(Standing, from left) Rong Tong, associate professor in chemical engineering; Wenjun "Rebecca" Cai, associate professor in materials science and engineering; Eungyo Jang; and Ziyu Huo gather around Liqian Niu (seated), who is working with a Luminex 200 machine used to analyze tumor cytokine levels.
(Standing, from left) Rong Tong, associate professor in chemical engineering; Wenjun “Rebecca” Cai, associate professor in materials science and engineering; Eungyo Jang; and Ziyu Huo gather around Liqian Niu (seated), who is working with a Luminex 200 machine used to analyze tumor cytokine levels. [Photo by Hailey Wade for Virginia Tech.]
Tong, Cai, and colleagues wanted to find a balance between killing cancer cells in the body and sparing healthy cells. “Scientists determined a while ago that cytokines can be used to activate and fight against tumors, but they didn’t know how to localize them inside the tumor while not exposing toxicity to the rest of the body,” said Tong. “Chemical engineers can look at this from an engineering approach and use their knowledge to help refine and elevate the effectiveness of the cytokines so they can work inside the body effectively.”

To accomplish this goal, the researchers used their expertise to create specialized microparticles particles with distinctive sizes that help determine where the drug is going. These microparticles are designed to stay within the tumor environment after being injected into the body. Cai and her students worked on measuring these particles’ surface properties.

“In the field of materials science and engineering, we study the surface chemistry and mechanical behavior of materials, such as the specialized particle created for this project,” Cai said. “Surface engineering and characterization, along with particle size, play important roles in controlled drug delivery, ensuring prolonged drug presence and sustained therapeutic effectiveness.”

To ensure successful drug delivery, Tong and his chemical engineering students designed a novel strategy that anchors cytokines to these new microparticles, limiting the harm of cytokines to healthy cells, but still allows the particle-anchored cytokines to jump-start immune systems and recruit immune cells to attack cancer cells. Their strategy involved noncovalently anchoring commercially available Fc-fused cytokines to the surface of particles decorated with Fc-binding peptide.

Preclinical studies showed that particle-anchored cytokines accumulated in tumors, while showing minimal systemic toxicity in mice. “Injection of these particles into tumors resulted in prolonged tumor retention of cytokines with minimal systemic toxicity, and the retention time was particle size dependent,” the investigators stated. They then combined the cytokine microparticles with commercially available, FDA-approved checkpoint blockade antibodies to effectively reactivate the tumor immune cells that have been silenced so they can fight back the cancer cells.

Studies in various syngeneic tumor models and genetically engineered murine tumor models confirmed that intratumoral administration of checkpoint antibodies in combination with the particle-anchored IL-12 (P-IL-12) and IL-15 (P-IL-15) cytokines effectively eliminated tumors, amplifying antitumor immunity in immune-excluded tumors, including in difficult-to-treat genetically engineered mouse models. “One major factor in resistance to checkpoint inhibitors is the lack of tumor-infiltrating immune cells such as T cells and NK cells in tumors,” the investigators pointed out. “In this study, we demonstrated that the prolonged presence of IL-12 and IL-15 in tumors induced a robust influx of both CD8+T cells and NK cells … Our results suggest that IL-12 and IL-15 signaling could help reinvigorate exhausted CD8+ T cells toward ICB antibody–responsive states, thereby generating durable antitumor immunity and immune memory.”

Tong stated, “Our strategy not only minimizes cytokine-induced harm to healthy cells, but also prolongs cytokine retention within the tumor. This helps facilitate the recruitment of immune cells for targeted tumor attack.” The team further reported, “‘Rechallenge studies in a 4T1 breast tumor model demonstrated that our combination therapy generated durable antitumor immune memory to control disseminated metastases. Note that the syngeneic murine tumor models studied here (4T1 and B16F10) were selected because of their resistance to ICB antibody treatment.” 4T1 is a mouse model of breast cancer, and B16f10 a model of melanoma.

The researchers hope that their impact on immunotherapy treatment is part of a greater movement toward cancer treatment approaches that are harmless to healthy cells. “Given the readily accessibility of Fc cytokines, our formulation provides a simple, general strategy for delivering potent immunostimulatory cytokines to induce durable antitumor immunity with minimal systemic toxicity,” they stated. “Our formulation strategy renders a safe and tumor-agnostic approach that uncouples cytokines’ immunostimulatory properties from their systemic toxicities for potential clinical application.”

The new approach of attaching cytokines to particles also could be used in the future to deliver other types of immunostimulatory drugs, according to the team. “Researchers are still looking for safer and more effective cancer treatments,” said Tong. “This motivation is what drives us to develop new technologies in the field. The whole class of drugs that are employed to jump-start the immune system to fight cancer cells has largely not yet succeeded. Our goal is to create novel solutions that allow researchers to test these drugs with existing FDA-approved therapeutics, ensuring both safety and enhanced efficacy.”

Cai said the nature of cancer treatment research requires expertise across engineering disciplines. “I view this project as a perfect marriage between chemical engineering and materials science. The former focuses on the synthesis and drug delivery part, the latter on applying advanced materials characterization. This collaboration not only accelerates immunotherapy research, but also has the ability to transform cancer treatment.”

Previous articleHow Biotechs Are Plugging AI into Drug Discovery Problems
Next articleAssembloids Model Patient-Specific Lung Cancers with Tumor Microenvironments