Turning a tumor's "shield" into a weapon against itself

According to Peter Insio Wang, tumor cells are “cunning.” They have sinister ways of evading human immune responses that battle these cancerous invaders. Tumor cells express programmed death ligand 1 (PD-L1) molecules, which act as a protective shield that suppresses our immune cells, creating an obstacle to targeted cancer immunotherapies.

Wang, the Alfred E. Mann Chair in Biomedical Engineering and the Dwight K. And Hildagard E. Baum Chair in Biomedical Engineering, leads a laboratory dedicated to pioneering research into engineered immunotherapies that harness the human immune system to build a future arsenal in the fight against cancer.

Wang's lab researchers have developed a new approach that turns a tumor cell's insidious defense mechanisms against itself, turning these "shield" molecules into targets for Wang's lab's chimeric antigen receptor (CAR) T cells, which are programmed to attack the cancer. p>

The work, conducted by Wang's lab postdoctoral fellow Lingshan Zhu, along with Wang, postdoctoral fellow Longwei Liu, and their co-authors, was published in the journal ACS Nano.

CAR T-cell therapy is a revolutionary cancer treatment in which T-cells, a type of white blood cell, are removed from the patient and equipped with a unique chimeric antigen receptor (CAR). CAR binds to antigens associated with cancer cells, directing T cells to destroy cancer cells.

Wang's lab's latest work is a designed monobody for CAR T cells, which the team calls PDbody, that binds to the PD-L1 protein on a cancer cell, allowing the CAR to recognize the tumor cell and block its defenses.

"Imagine that the CAR is a real car. You have an engine and gasoline. But you also have a brake. Essentially, the engine and gasoline push the CAR T to move forward and destroy the tumor. But PD-L1 acts as a brake, which stops him," Wang said.

In this work, Zhu, Liu, Wang and team engineered T cells to block this inhibitory “braking” mechanism and turn the PD-L1 molecule into a target for killing.

"This PDbody-CAR chimeric molecule can lead our CAR T to attack, recognize and destroy the tumor. At the same time, it will block and prevent the tumor cell from stopping the CAR T attack. Thus, our CAR T will be more powerful," said Wang.

CAR T-cell therapy is most effective for “liquid” cancers such as leukemia. The goal for the researchers was to develop advanced CAR T cells that can distinguish between cancer cells and healthy cells.

Wang's lab is exploring ways to target the technology to tumors so that CAR T cells are activated at the tumor site without affecting healthy tissue.

In this work, the team focused on a highly invasive form of breast cancer that expresses the protein PD-L1. However, PD-L1 is also expressed by other cell types. Therefore, the researchers looked at the unique tumor microenvironment—the cells and matrices immediately surrounding the tumor—to ensure that their designed PDbody would bind more specifically to cancer cells.

"We know that the pH in the tumor microenvironment is relatively low—it's a little acidic," Zhu said. "So we wanted our PDbody to have better binding ability in an acidic microenvironment, which will help our PDbody distinguish tumor cells from other surrounding cells."

To improve treatment precision, the team used a proprietary genetic gate system called SynNotch, which ensures that CAR T cells with a PDbody only attack cancer cells that express a different protein known as CD19, reducing the risk of damage to healthy cells.

"Simply put, T cells will only be activated at the tumor site thanks to this SynNotch gating system," Zhu said. "Not only is the pH more acidic, but the surface of the tumor cell will determine whether the T cell is activated, giving us two levels of control."

Zhu noted that the team used a mouse model, and the results showed that the SynNotch gating system directs CAR T cells with a PDbody to activate only at the tumor site, killing tumor cells and remaining safe for other parts of the animal.

Evolution-inspired process to create the PDbody

The team used computational methods and took inspiration from the process of evolution to create their custom PDbodies. Directed evolution is a process used in biomedical engineering to mimic the process of natural selection in a laboratory setting.

The researchers created a directed evolution platform with a giant library of iterations of their designed protein to discover which version might be most effective.

"We needed to create something that would recognize PD-L1 on the tumor surface," Wang said.

"Using directed evolution, we selected a large number of different monobody mutations to select which one would bind to PD-L1. The selected version has these features that can not only recognize tumor PD-L1, but also block the inhibitory mechanism, which it has, and then direct the CAR T cell to the surface of the tumor to attack and destroy the tumor cells."

"Imagine if you wanted to find a very specific fish in the ocean - it would be really difficult," Liu said. "But now with the directed evolution platform we've developed, we have a way to target these specific proteins with the desired function."

The research team is now exploring how to optimize the proteins to create even more precise and effective CAR T cells before moving into clinical applications. This also includes integrating the proteins with Wang's lab's breakthrough focused ultrasound applications to remotely control CAR T cells so they activate only at tumor sites.

"We now have all these genetic tools to manipulate, control, and program these immune cells to have as much power and function as they can," Wang said. "We're hoping to create new ways to direct their function for particularly challenging solid tumor treatments."