Most CAR T-cell therapies look for antigens specific to cancer cells. A new approach instead targets the environment around the tumor, using unusually small antibodies made naturally by alpacas, camels and llamas. Using this approach in mouse models, investigators successfully curbed melanoma and colon cancer - solid tumors that currently can't be treated with CAR T-cell therapy.

In 1989, a couple of biology students at Free University of Brussels had been asked to test frozen camels’ blood serum, and discovered a formerly unknown type of antibody. It was an undersized version of the molecules, made up of two heavy chains only and naturally devoid of light chains. As eventually reported, this specific kind of antibodies, heavy chains antibodies, were found not only camels, but also in llamas, alpacas and, surprisingly, sharks and other cartilaginous fish.  

30 years later, researchers at Boston Children's Hospital and Massachusetts Institute of Technology show that these heavy chain antibodies, further reduced in size to create so-called nanobodies, may also assist in solving a problem in the cancer field: making CAR T-cell therapies work in solid tumors.

Very promising in treatment for blood cancer, chimeric antigen receptor (CAR) T-cell therapy genetically engineers a patient's own T cells to make them better at attacking cancer cells. Nowadays, The Dana-Farber/Boston Children's Cancer and Blood Disorders Center is using CAR T-cell therapy for relapsed acute lymphocytic leukemia (ALL), for instance.



But CAR T cells haven't showed great results at eradication of solid tumors. It's been hard to locate most cancer-specific proteins on solid tumors that could serve as safe targets. Solid tumors are also protected by an extracellular matrix, a supportive net of proteins that acts as a barrier, in addition to immunosuppressive molecules that weaken the T-cell attack.

That's where nanobodies step in. Almost two decades the technology belonged and mostly remained in the hands of Belgian researches team. However, that changed in 2013, when patent expired.

At that time, many people got interested in Nanobodies ’unique properties.One useful feature is their enhanced targeting abilities. Ploegh and his team at Boston Children's, in collaboration with Noo Jalikhani, PhD, and Richard Hynes, PhD at MIT's Koch Institute for Integrative Cancer Research, have harnessed nanobodies to carry imaging agents, allowing particular visualization of metastatic cancers.

The Hynes team targeted the nanobodies to the tumors' extracellular matrix, or ECM -- aiming imaging agents not at the cancer cells themselves, but at the environment that surrounds them. Such markers are common to many tumors, but don't typically appear on normal cells.

"Our lab and the Hynes lab are among the few actively pursuing this approach of targeting the tumor micro-environment," says Ploegh. "Most labs are looking for tumor-specific antigens." Ploegh's lab took this idea to CAR T-cell therapy. His group, including members of the Hynes lab, took aim at the very factors that make solid tumors difficult to treat.

The CAR T cells they created have been studded with nanobodies that recognize specific proteins in the tumor environment, bearing signals directing them to kill any cell they bound to. One protein, EIIIB, a variant of fibronectin, is found only on newly formed blood vessels that supply tumors with nutrients. Another, PD-L1, is an immunosuppressive protein that most cancers use to silence approaching T cells.

Biochemist Jessica Ingram, PhD of the Dana-Farber Cancer Institute, Ploegh's partner and a coauthor on the paper, led the manufacturing pipeline. She would drive to Amherst, Mass., to gather T cells from two alpacas, Bryson and Sanchez, inject them with the antigen of interest and harvest their blood for further processing back in Boston to generate nanobodies.

Tested in two separate melanoma mouse models, as well as a colon adenocarcinoma model in mice, the nanobody-based CAR T cells killed tumor cells, significantly slowed tumor growth and improved the animals' survival, without readily apparent side effects.

Ploegh thinks that the engineered T cells work through a combination of factors. They caused damage to tumor tissue, which tends to stimulate inflammatory immune responses. Targeting EIIIB may damage blood vessels in a way that decreases blood supply to tumors, while making them more permeable to cancer drugs.

"If you destroy the local blood supply and cause vascular leakage, you could perhaps improve the delivery of other things that might have a harder time getting in," says Ploegh. "I think we should look at this as part of a combination therapy."

Ploegh thinks his team's approach could be useful for eliminating of many solid tumors. He's particularly interested in testing nanobody-based CAR T cells in models of pancreatic cancer and cholangiocarcinoma, a bile duct cancer from which Ingram passed away in 2018.

The technology itself can be pushed even further, says Ploegh.

"Nanobodies could potentially carry a cytokine to boost the immune response to the tumor, toxic molecules that kill tumor and radioisotopes to irradiate the tumor at close range," he says. "CAR T cells are the battering ram that would come in to open the door; the other elements would finish the job. In theory, you could equip a single T cell with multiple chimeric antigen receptors and achieve even more precision. That's something we would like to pursue."

See the paper for details on authors and funders.

Source: Boston Children's Hospital's science and clinical innovation blog

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