Most CAR-T cell therapies require targeting cancer cell-specific antigens. Now, there is a new way to target the environment around the tumor, which comes from nanobodies naturally produced by free alpacas, camels and llamas. Using this method in mouse models, the researchers successfully inhibited melanoma and colon cancer that currently cannot be treated with CAR-T cell therapy.

Bryson and Sanchez, two alpacas produced nanobodies. (Photo: Boston Children’s Hospital)

In 1989, two undergraduates of Free University of Brussels stumbled upon an unknown antibody while testing camel’s frozen serum. It is a miniaturized version of human antibodies and consists of only two heavy chains, not two light chains and two heavy chains. Their final report says the presence of the antibody has been confirmed not only in camels, but also in alpacas and alpacas. Thirty years later, in the PNAS, researchers at Boston Children’s Hospital and the Massachusetts Institute of Technology showed that further reduction of these mini antibodies could create so-called nanobodies. It may help solve the problem in the field of cancer: making CAR-T cell therapy work in solid tumors.

Chimeric antigen receptor (CAR) T cell therapy through genetic engineering to modify the patient’s own T cells, so that it can better attack cancer cells, which shows a great development prospect for blood tumors. For example, Dana-Farber / Boston Children’s Cancer and Blood Disease Center is currently using CAR-T cell therapy to treat recurrent acute lymphoblastic leukemia (ALL). However, CAR-T cells still face great challenges in eliminating solid tumors, and it is difficult to find tumor-specific proteins that can be used as safe targets in solid tumors. At the same time, solid tumors are also protected by extracellular matrix, which is a supporting network composed of proteins and acts as a barrier. At the same time, immunosuppressive molecules can also weaken the attack of T cells.

Rethinking CAR-T cells

For two decades, antibody patents have been largely held by Belgium. But that changed after the patent expired in 2013. Dr. Hidde Ploegh, a senior researcher and immunologist at Boston Children and PNAS Research, said: “A lot of people are aware of this field and are beginning to recognize the unique nature of nanobodies. “A useful feature is that they enhance their positioning capabilities. Ploegh of Boston Children’s Hospital and his team, in collaboration with Dr. Noo Jalikhani and Dr. Richard Hynes of the Koch Comprehensive Cancer Institute at the Massachusetts Institute of Technology, have used nanoparticles to carry imaging agents to accurately visualize metastatic cancer.

The Hynes team maked nanobodies target the tumor’s extracellular matrix (ECM), the environment around the cancer, rather than the cancer cell itself. This marker is common in many tumors, but usually does not appear in normal cells. “our laboratory and Hynes laboratory are one of the few laboratories that actively study this method of targeting the tumor microenvironment, and most laboratories are looking for tumor-specific antigens,” Ploegh said.

Targeted tumor microenvironment

Ploegh and his team targeted factors that make solid tumors difficult to treat and applied the idea to CAR-T cell therapy. They created CAR-T cells filled with nanosomes that recognize specific proteins in the tumor environment and carry signals instructing them to kill any cells that bind to them. One of the proteins, EIIIB, is a variant of fibronectin, which exists only in newly formed blood vessels that provide nutrition for tumors. The other is PD-L1, an immunosuppressive protein that most cancers use to inhibit close T cells.

Doctor of the Dana-Farber Cancer Institute, Jessica Ingram, drove to Amherst, Massachusetts, to collect T cells from two alpaca Bryson and Sanchez, inject them with interested antigens and collect blood. Further processed in Boston to produce nanobodies.

Treatment of melanoma and colon cancer

It was tested in two independent melanoma mouse models and a colon adenocarcinoma mouse model. CAR-T cells based on nanobodies kill tumor cells, significantly slow down tumor growth, improve the survival rate of animals, and have no obvious side effects.

Ploegh believes that engineered T cells work together through a variety of factors. They cause damage to tumor tissue, which often stimulates the inflammatory immune response. Targeted EIIIB may damage blood vessels in a way that reduces tumor blood supply while making them more permeable to cancer drugs. “If you disrupt the local blood supply and cause vascular leakage, it may improve the delivery of other more inaccessible things, and I think we should see this as part of a combination therapy,” Ploegh said.

Future development direction

Ploegh thinks his team’s approach may be useful for many solid tumors. He was particularly interested in testing CAR-T cells based on nanobodies in pancreatic and cholangiocarcinoma models. “Nanobodies may carry a cytokine to enhance the immune response to tumors, such as toxic molecules that kill tumors and radioisotopes that irradiate tumors at close range,” Ploegh said. “CAR-T cells are the vanguard of breaking through the gates and the other elements then complete their mission. In theory, you can equip a T cell with multiple chimeric antigen receptors and achieve higher accuracy, which is what we want to pursue.”