Small targets, big results: EVA1-directed CAR-T cells show promise for solid tumors

Scientists at Nagoya University have designed immune cells that eliminate solid tumors in laboratory mice by targeting EVA1, a small protein rich in cancer cells but rarely present in healthy tissues.

Modified CAR-T cells represent potential novel therapeutic approaches for lung, pancreatic, and liver cancers that have been proven difficult to treat with existing treatments.

This study, published in the Journal for Immunotherapy for Cancer, shows how small size of EVA1 (only 128 amino acids) is indeed an ideal target for immunotherapy. Unlike large-scale proteins targeted previous CAR-T approaches, the compact structure of EVA1 allows immune cells to form closer connections with cancer cells and improve treatment efficacy.

Small size, big impact

CAR-T therapy works by genetically altering a patient’s T cells to recognize specific markers of cancer cells. Despite its huge success against blood cancer, this approach struggles with solid tumors due to the challenge of finding the right target protein.

A small footprint on the cell surface of EVA1 may solve this problem. Researchers have discovered that when Car-T cells connect to EVA1, they form what scientists call “immune synapses.”

The team tested 16 different versions of humanized antibodies to find the most effective design. Their optimal versions were used using a short connector piece between immune cells and cancer cells in combination with specific protein components that enhance the T cell response.

Promising test results

In mouse studies, EVA1 targeting CAR-T cells completely eliminated tumors using relatively small doses. This is 1-2 million cells compared to 3-10 million cells required for other solid tumor CAR-T treatments. This approach worked for both lung cancer and pancreatic cancer models.

What makes EVA1 particularly attractive is its expression patterns. Protein appears at high concentrations in malignant tumors of the lung, pancreas, and liver, but exhibits much lower levels in most normal cells. This selectivity can reduce the side effects that can cause current cancer treatments.

This study revealed that EVA1 requires a minimum threshold of approximately 15,000-20,000 protein copies per cell to trigger an immune response. Normal esophageal cells, which had only 7,500 copies, are unable to activate modified T cells, suggesting that treatment does not spare healthy tissue.

Address safety concerns

One potential complication appeared during the test. EVA1 appears in small amounts in monocytes, a type of white blood cell. However, modified immune cells are only activated when cells with high levels of EVA1 are encountered, almost neglecting small amounts of leukocytes.

“These findings are an important step towards new treatment options for difficult-to-treat cancers. Patients with different types of tumors expressing the EVA1 protein could be useful in this approach,” explained Dr. Seitaro Terakura, Graduate School of Nagoya University.

The researchers addressed potential immune rejection by starting with antibodies from mice and modifying them to resemble human antibodies. This humanization process should prevent the patient’s immune system from attacking the treatment itself.

Important design discoveries

This study uncovered important technical insights that could improve future CAR-T therapies. Contrary to expectations, the shorter connecting domains between immune cells and cancer cells were found to be more effective than the longer ones in living animal tests despite the weaker response in the laboratory dish.

The researchers found that EVA1 does not shed from the cell surface like several other cancer proteins. Once the protein breaks out of cancer cells, treatment can be intercepted before it reaches the intended target.

Furthermore, EVA1 lacks the protease recognition sequence, namely molecular scissors that loosen proteins from the cell membrane. This stability can increase the durability of the treatment.

The key benefits are:

• The smaller the protein size, the closer the connections of immune cells
• High expression in cancer cells, low in normal cells
• Smaller CAR-T cell doses are effective
• Proteins do not shed from the surface of cancer cells
• Target multiple cancer types successfully

Move towards human exams

The next important step is to check the safety of additional models before proceeding with human testing. The researchers will develop a mouse version of the treatment to thoroughly assess potential side effects.

“Our team will focus on verifying the safety of human treatments before moving on to clinical trials,” Terrakula noted. “To determine whether EVA1 Car-T can be safely administered to humans, we are currently generating mouse Car-T that recognizes mouse EVA1.”

Researchers need to demonstrate that mouse versions do not cause severe toxicity when targeting EVA1 expressed in normal mouse tissues. This safety data is essential for regulatory approval of human testing.

“Following the accumulation of such data, we hope to work with businesses and others to move towards clinical applications,” Tirakula added.

The broader meaning

EVA1 research provides insights that could benefit Car-T development beyond this specific goal. The finding that protein size affects immune synapse formation suggests that molecular dimensions should be considered when selecting future targets.

This study also demonstrates how mouse antibodies can reduce immune rejection while maintaining effectiveness. This is an important consideration for treating patients with an intact immune system, unlike severely immunocompromised individuals who are receiving regular CAR-T therapy.

For patients facing lung, pancreatic, or liver cancer, EVA1 CAR-T therapy may ultimately offer new hope. These types of cancer have historically been proven difficult to treat and often carry poor prognosis, making new approaches particularly valuable.

This study represents a step towards expanding the reach of CAR-T therapy from hematological cancers that have achieved significant success in solid tumors, which account for the majority of cancer deaths around the world.

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