Treatment with chimeric antigen receptor (CAR) T-cells, which refer to T-cells that are genetically modified to express chimeric antigen receptors, have revolutionized treatment of B-cell malignancies. Since their first introduction in the clinic, they have been shown to have unprecedented efficacy in B cell malignancies, most notably in B cell acute lymphoblastic leukemia (B-ALL) with up to a 90% complete remission rate using anti-CD19 CAR-T cells.

For patients diagnosed with some forms of hematological malignancies, treatment with a CAR T-cells therapy may offer a last chance of overcoming cancer. However, CAR T-cell therapy is still faced with numerous challenges. Hence, enhancing the efficacy of engineered T-cells without compromising their safety is warranted.

Colorized scanning electron micrograph of a T lymphocyte. Photo courtesy: © 2022 NIAID. Used with permission.

CAR T-cell therapy
Treatment with CAR T-cell therapy involves taking T-cells from the patient’s blood and adding artificial receptors – the CARs – to them in the lab. As the guards of our immune system, T-cells are on “permanent patrol” in our blood vessels and tissues, where they hunt down foreign structures. Equipped with CARs, T cells can also detect very specific surface structures on cancer cells. Once the CAR T-cells are returned to the patient by infusion, they circulate in the body as a kind of living drug that can bind to very specific tumor cells and destroy them.

The engineered immune cells remain in the body permanently and multiply. If the cancer flares up again, they’ll go back into action. That is, at least, the theory. But in practice, patients still relapse. This is because the tumor cells can outwit the CAR T-cells by producing more of a protein called estrogen receptor-binding fragment- associated antigen 9 (EBAG9) which inhibits the release of cytolytic enzymes from cytotoxic T lymphocytes.

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In their research, Armin Rehm, MD, Uta Höpken, Ph.D, researchers at Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC) in Berlin, Germany, have identified a mechanism that tumor cells use to dodge the body’s immune response and demonstrated that shutting down the EBAG9 gene in mice led to a sustained increase in the immune response to cancer. The mice also developed more T-memory cells. These cells are part of our immunological memory, which allows our immune system to respond better to a cancer antigen after encountering it previously.[1]

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“In many cases, tumor cells read the EBAG9 gene especially often. The cells then produce a protein that protects them. But EBAG9 also influences the cells of the immune system because T cells produce it too. In T cells, EBAG9 inhibits the secretion of enzymes that act as poison to kill tumor cells,” says Rehm.

Further research and development
In ongoing research the scientists also demonstrated their key findings in vitro, in human CAR T-cells. Writing in Molecular Therapy, the team says that this is the decisive step on the road to therapeutic use.[2]

“Shutting down EBAG9 allows the body to eradicate tumor cells earlier and more radically. As well as achieving longer-lasting therapeutic success, this could also create a real chance of cure,” says Rehm noted

Releasing the brakes
Following the discovery of the EBAG9 gene, researchers recognized that it played an important role in cancer. But it took a long time to identify what that role actually was. When the research team at Max Delbrück Center for Molecular Medicine started working on it in 2009, they found that mice without the gene dealt with bacterial and viral infections much better than mice with the gene, and that they formed more T-memory cells, which are of particular interest in tumor biology.

Then in 2015, lead author Anthea Wirges Ph.D., succeeded in curbing synthesis of the EBAG9 protein using microRNA. For the latest study, she used microRNA to cultivate “EBAG9-silenced” CAR T cells with different human leukemia or lymphoma cells. Just like in the mouse model, the silencing reduced tumor growth much more. Relapses also only developed much later.

“By released this brake we shut down the EBAG9 gene,” Wirges noted. “This meant we could stop EBAG9 being produced in the T-cells and strengthen the immune response to cancer for the long term,” Wirges said.

“Furthermore, releasing the EBAG9 brake allows the genetically engineered T-cells to release more cytotoxic substances. However, they don’t cause the strong cytokine storm that is typically a side effect of CAR therapy,” Wirges explained.

In fact, the risk is minimized because fewer cells are used. “Switching off the immune brake works across the board. We can do it with every CAR T cell that we produce – regardless of which type of blood cancer it targets,” she added.

The next step
However, the first-line therapy for blood cancer will remain chemotherapy combined with conventional antibody therapy, as many patients respond very well to this.

“CAR T-cell therapy only comes into play if the cancer returns. It’s very expensive because it’s an individual cellular product for a single person,” said Höpken.  But a single treatment with that product can save a life.

The EBAG9 work shows how important perseverance and patience are for researchers. Wirges was motivated by the prospect of her work having a real chance of clinical application.

“Projects like this allow you to get to grips with a technique in basic research and then apply everything in translational research – right up to toxicological screening for the regulatory processes,” Rehm added.

Their project has now reached this last stage and in November 2022 the researchers are expected to present their concept to the Paul Ehrlich Institute, Germany’s biologics approval agency.

Based on their ongoing research the scientists are convinced that releasing the EBAG9 brake is highly effective and doesn’t cause any more side effects than conventional CAR T-therapy.

“We now need the clinicians and a partner for financing the clinical studies,” said Rehm.

“If everything goes well, the therapy using EBAG9-silenced CAR T-cells could be available to patients in as little as two years’ time,” he concluded.

Reference
[1] Rehm A, Wirges A, Hoser D, Fischer C, Herda S, Gerlach K, Sauer S, Willimsky G, Höpken UE. EBAG9 controls CD8+ T cell memory formation responding to tumor challenge in mice. JCI Insight. 2022 Jun 8;7(11):e155534. doi: 10.1172/jci.insight.155534. PMID: 35482418; PMCID: PMC9220939.
[2] Wirges A, Bunse M, Joedicke JJ, Blanc E, Gudipati V, Moles MW, Shiku H, Beule D, Huppa JB, Höpken UE, Rehm A. EBAG9-silencing exerts an immune checkpoint function without aggravating adverse effects. Mol Ther. 2022 Jul 11:S1525-0016(22)00430-0. doi: 10.1016/j.ymthe.2022.07.009. Epub ahead of print. PMID: 35821635.

Featured image: Armin Rehm (left) and Uta Höpken (right). Photo courtesy: © 2022 Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC)/Anyess von Bock. Used with permission.

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