Researchers from North Carolina State University (NC State) and the University of North Carolina (UNC) at Chapel Hill have developed a “bioinstructive” implantable scaffold that manufactures and releases CAR T cells directly in the body, to attack cancerous tumors. In a proof-of-concept study in lymphoma-carrying mice, the researchers found that treatment using the “multifunctional alginate scaffold for T cell engineering and release” (MASTER) implants was faster and more effective than conventional CAR T cell cancer therapy. The new platform technology also allowed the researchers to reduce the CAR T cell processing time from weeks, to just a day.

Close-up of scanning electron microscopy of the microstructure of MASTER scaffolds showing widespread porosity, which is critical to the scaffold’s function. [Cellular and Molecular Imaging Facility, NC State University]
First author Pritha Agarwalla, PhD, a postdoc researcher in the joint biomedical engineering department at NC State and UNC, and colleagues reported their work in a paper in Nature Biotechnology, titled “Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR T cells,” in which they concluded, “MASTER promises to transform CAR T cell therapy by fast-tracking manufacture and potentially reducing the complexity and resources needed for provision of this type of therapy.”

Immune system T cells are tasked with identifying and destroying cells in the body that have become infected with an invading pathogen. CAR T cells are T cells that have been engineered specifically to identify cancer cells and destroy them. CAR T cells are already in clinical use for treating lymphomas, and many clinical trials are underway to evaluate the use of CAR T cell treatments against other forms of cancer.

“CAR T cell therapy has demonstrated unprecedented success against CD19-expressing B cell malignancies,” the authors further explained, and this success has resulted in two FDA approvals, and inspired “hundreds of ongoing clinical trials.” However, despite the revolutionary potential of CAR T cell therapies to treat human malignancies, the complex procedures and costs associated with producing clinical-grade CAR T cells is a major obstacle to widespread clinical use. “… CAR T cell therapies for B cell malignancies are limited by lengthy, costly, and labor-intensive ex vivo manufacturing procedures …” the team continued. “The complete manufacturing process can cost up to a half-million dollars and can take several weeks. This delay is problematic because the aggressiveness of many cancers might not allow sufficient time to complete the production … The costs and challenges associated with producing CAR T cells have driven research into improved manufacturing methods.”

“A major drawback to CAR T cell treatment is that it is tremendously expensive—hundreds of thousands of dollars per dose,” added Yevgeny Brudno, PhD, corresponding author of the study and assistant professor in the joint biomedical engineering department at NC State and UNC. “Due to its cost, many people are shut out from this treatment. One reason for the high cost is that the manufacturing process is complex, time-consuming, and has to be tailored to each cancer patient individually,” Brudno added. “We wanted to address challenges in CAR T treatment related to both manufacturing time and cost.”

“Reducing the manufacturing time is even more critical for patients with rapidly progressing disease,” commented Agarwalla. The researchers created MASTER to tackle this challenge. Their work was carried out in partnership with Gianpietro Dotti, PhD, professor in the department of microbiology and immunology and co-leader of the immunology program at the Lineberger Cancer Center at UNC; and Frances Ligler, PhD, a professor of biomedical engineering at Texas A&M University.

Understanding how CAR T cells are produced helps to understand how MASTER works. To generate CAR T cells clinicians first isolate T cells from patients, and they are transported to manufacturing. The T cells are then activated using antibodies over several days, to prepare them for reprogramming. Viruses are then used to introduce the CAR gene into the activated T cells, reprograming them into CAR T cells that target cancer cells. Researchers then add factors to stimulate the CAR- T cells to proliferate, expanding their number. This whole process can take weeks, and it’s only then that the cells can be returned to the hospital and infused into the patient’s bloodstream.

View of the entire MASTER scaffold by scanning electron microscopy. [James Weaver, Harvard University]
“Our MASTER technology takes the cumbersome and time-consuming activation, reprogramming, and expansion steps and performs them inside the patient,” Agarwalla explained. “This transforms the multi-week process into a single-day procedure.”

MASTER is a biocompatible, sponge-like material with the look and feel of a mini marshmallow. To begin treatment, researchers isolate T cells from the patient and mix these naïve (non-activated) T cells with the engineered virus. The mixture is poured on top of the MASTER, into which it is absorbed. MASTER is decorated with the antibodies that activate the T cells, so the cell activation process begins almost immediately. The MASTER scaffold is then surgically implanted into the patient.

After implantation, the cellular activation process continues. As the T cells become activated, they begin responding to the modified viruses, which reprogram them into CAR T cells. “The large pores and sponge-like nature of the MASTER material bring the virus and cells close together, which facilitates cellular genetic reprogramming,” Agarwalla commented. And as the authors noted, “MASTER can be directly loaded with patient-derived T cells and viral particles encoding the CAR and implanted on the same day to generate CAR T cells in vivo.”

The MASTER scaffold material is also impregnated with factors called interleukins that foster cell proliferation. After implantation, these interleukins begin to leach out, promoting rapid proliferation of the CAR-T cells. “Engineering the material so that it is dry and absorbs this combination of T cells and virus is critically important,” Brudno said. “If you try to do this by applying T cells and virus to a wet MASTER, it just doesn’t work.”

In their paper, the authors summarized the key features of the MASTER platform: “MASTER is designed to (1) host T cells and viral particles; (2) stimulate T-cell activation and proliferation; (3) promote T-cell transduction; and (4) locally expand CAR T cells and (5) sustainably release fully functional CAR T cells to control tumor growth.”

For their reported studies, the researchers tested the MASTER technology in lymphoma-bearing mice. One group of animals was treated with CAR T cells that were created and delivered using MASTER. A second group was treated with CAR T cells that were created conventionally and delivered intravenously. The results in these two groups were then compared with those of a control group receiving non-engineered T cells.

“Our technology performed very well,” Brudno said. “It would take at least two weeks to create CAR T cells from naïve T cells for clinical use. We were able to introduce the MASTER into a mouse within hours of isolating naïve T cells.”

In addition, since cells are implanted within hours of isolation, the minimal manipulation creates healthier cells that exhibit fewer markers associated with poor anticancer performance in CAR T cells. Specifically, the MASTER technique results in cells that are less differentiated, which translates to better sustainability in the body and more anticancer potency. In addition, the cells display fewer markers of T-cell exhaustion, which is defined by poor T-cell function.

“The end result is that the mice that received CAR T cell treatment via MASTER were far better at fighting off tumors than mice that received conventional CAR-T cell treatment,” Agarwalla noted.

The improvement in anticancer efficacy was especially pronounced over the long term, when mice were faced with a recurrence of lymphoma. Taken together, the authors said, their results “ … demonstrate that CAR T cells produced using MASTER were equally functional to conventional CAR T cells in controlling tumor growth but had better expansion and persistence. Notably, MASTER substantially reduced the time, complexity and cost of CAR T cell production.”

“The MASTER technology was very promising in liquid tumors, such as lymphomas, but we are especially eager to see how MASTER performs against solid tumors—including pancreatic cancer and brain tumors,” Brudno commented. “We’re working with an industry partner to commercialize the technology, but there’s still a lot of work to be done before it becomes clinically available. Further work to establish the safety and robustness of this technology in animal models will be necessary before we can begin exploring clinical trials involving human patients.”

While it’s impossible to estimate what the cost of MASTER treatment might be if it is eventually approved for clinical use, Brudno says he’s optimistic that it would be substantially less expensive than existing CAR T treatment options.

The MASTER concept may have utility in areas other than cancer, the researchers suggested. “Beyond its potential for cancer therapy, the MASTER technology might inspire new treatments harnessing the capacity for reprogramming and release of therapeutic cells.” Brudno stated, “We’re also exploring opportunities with other industry partners for taking the fundamental concepts of MASTER and applying them for use in regenerative medicine and in treating autoimmune disease.”

“I feel like we’re just scratching the surface of what’s possible here,” Agarwalla concluded.

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