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In a world first, researchers from Penn Medicine have used genetically modified T cells to target and ablate fibroblasts that contribute to the development of heart disease.



What is CAR-T cell therapy?



CAR-T cell therapy is a relatively novel form of immunotherapy that is being used to treat certain types of cancer, primarily blood cancers. A form of adaptive cell transfer (ACT), CAR-T cell therapy involves genetically modifying a patient's own T cells in the laboratory.



The T cells are extracted from the patient's blood, and the gene for a chimeric antigen receptor (CAR) is inserted. These receptors are "synthetic", meaning they do not exist naturally.



The CAR is expressed on the cell surface of the T cell and enables the immune cell to recognize specific antigens expressed in cancer cells. Thus, when the T cells are infused back into the patient, they target and kill the cancer cells.









Video credit: YouTube.





The world's first approved CAR-T cell therapy, Kymriah, was developed by Penn Medicine's Abramson Cancer Center.



The breakthrough use of CAR-T cell therapy in cancer treatment has encouraged scientists to explore its viability as a treatment option for other human diseases. In a new study published in Nature, Penn Medicine researchers have explored its potential for removing activated fibroblasts that contribute to the development of heart disease.



Heart disease – the leading cause of death in the United States



Approximately 610,000 people die of heart disease in the United States each year, accounting for one in every four deaths. It is the leading cause of death for both males and females.



Cardiac fibrosis is a contributing factor to the many different types of heart disease, caused by a proliferation of cardiac fibroblasts. Under normal conditions, cardiac fibroblasts provide structural support for the heart by depositing and maintaining extracellular matrix. When these cells are over-activated, often after cardiac injury, they deposit excess extracellular matrix in the cardiac muscle, causing thickening of the muscle and a loss of flexibility.1



Previous research has demonstrated that the removal of activated cardiac fibroblasts can restore heart function. Jonathan Epstein, MD, executive vice dean and professor of cardiovascular research at Penn Medicine tells us: "There is a lot of research using genetic strategies to ablate cells in animal models. There were two such studies that used a genetic model of diphtheria toxin ablation of cardiac fibroblasts in mice (Kaur et al. 2016, Kanisicak et al. 2016). These studies found that if the cardiac fibroblasts were ablated, cardiac fibrosis was reduced and heart function was restored. Obviously, we cannot use those types of strategies in humans."



Extending cell immunotherapy beyond cancer treatment



Instead, the researchers built on cell immunotherapy work targeting cancer cells to try and target pathologic cardiac fibroblasts utilizing a mouse model.2



The mouse model expresses an artificial antigen (OVA) on cardiac fibroblasts and is steadily infused with agents to mimic high blood pressure. When asked why they adopted this particular model, first author Haig Aghajanian, PhD, tells us "We chose this model because the mouse hearts get very fibrotic under these conditions, and it is a good model for the type of hypertensive heart disease seen in humans – sometimes called a “stiff heart.”



To selectively target the OVA proteins expressing cardiac fibroblasts, Aghajanian and colleagues treated one cohort of mice with engineered CD8+ T cells that express a T cell receptor against the OVA peptide. They found that four weeks post-treatment, the mice with the re-engineered T cells had significantly less cardiac fibrosis compared to the control groups.



Finding the perfect target



Next, the scientists endeavoured to identify a protein that was specifically expressed by activated fibroblasts – not an artificially expressed protein as used in the previous experiment. They could then look to program the genetically modified T cells to recognize and attack the fibroblasts.



This proved to be the greatest challenge in the study, as Epstein tells us: "One of the biggest challenges was making sure to choose the right antigen to target. We wanted to make sure that we only targeted the cells that were pathologic and damaging to heart function, and not healthy, beneficial cells in the heart or elsewhere in the body."



To find their target, the scientists used an RNA sequencing database, to analyze gene expression data from patients that had heart disease. They struck gold in the form of the fibroblast activation protein (FAP) – a cell surface glycoprotein.



FAP CAR-T cells were engineered and transferred into mice. Much to the joy of Aghajanian and colleagues, one month post-transfer, a significant reduction in cardiac fibroblasts was discovered in the treated mice, compared to control mice. Physiological measures also demonstrated improved diastolic and systolic function of the heart.



Clinical translation of the findings



The researchers note that additional research is required to confirm FAP as the optimal target and to reduce safety risks, but the initial results are promising, "When we looked at human hearts from patients with heart failure, we found expression of the same antigen (FAP) on the cardiac fibroblasts characteristic of diseased hearts, suggesting that our therapy may be translatable to humans – though much work will be needed to ensure both safety and efficacy in people," says Epstein.



"Our results are one example of how new knowledge about how to engineer the immune system offers new therapeutic options for many human diseases. Nearly every organ and tissue in the body can be afflicted with excessive fibrosis (examples include liver cirrhosis, arthritis, chronic kidney disease) and there are few if any therapies. It is exciting that a strategy of targeting fibrosis with CAR T cells is indeed possible," Aghajanian adds.



Next steps for the "Immuno Revolution"



The scientists believe that the immunotherapy approach created in their study may have implications beyond fibrotic heart disease and could be translated to numerous untreatable fibrotic diseases.



"We are encouraged by our results to date and would like to see it advance to the clinic as soon as safely possible. Before we pursue first-in-human clinical trials, we would like to test this strategy in a large animal model to ensure safety and efficacy. In addition, we are interested in looking at additional types of heart disease as well as other fibrotic diseases," concludes Epstein.



References:

1. Tallquist, Michelle D., and Jeffery D. Molkentin. 2017. Redefining the Identity of Cardiac Fibroblasts. Nature Reviews Cardiology. DOI: https://doi.org/10.1038/nrcardio.2017.57.

2. Aghajanian et al. 2019. Targeting Cardiac Fibrosis with Engineered T cells. Nature.