In vivo generated chimeric antigen receptor T cells reduce fibrosis and restore cardiac function in experimental heart failure

Comment on ‘CAR T cells produced in vivo to treat cardiac injury’ published in Science (https://doi.org/10.1126/science.abm0594). In the current study, Rurik et al.1 developed a novel immunotherapy strategy to reduce fibrosis and restore cardiac function in a mouse model of heart failure (HF). To this aim, T cells were temporarily reprogrammed to attack activated heart fibroblasts. Transient engineered chimeric antigen receptor (CAR) T cells were generated in vivo, inducing the T cells to produce their own CAR. Specifically, mRNA encoding a T-cell receptor targeting the fibroblast activation protein (FAP) was encapsulated within lipid nanoparticles (LNPs) that were covered with antibodies against CD5, a receptor naturally expressed by T cells (CD5/LNP-FAPCAR). CD5-targeted LNPs were shown to deliver their mRNA cargo to ∼83% of cultured murine T cells that expressed fibroblast activation protein chimeric antigen receptor (FAPCAR) after exposure to CD5/LNP-FAPCAR. FAPCAR expression peaked at 24 h and rapidly declined over the following days. In vitro, these LNP-generated CAR T cells were able to effectively kill FAP-expressing target cells in a dose-dependent manner. Gene transfer through targeted LNPs was also possible in human T cells with an efficiency ranging from 89 to 93%. The efficacy of in vivo reprogrammed CAR T cells was evaluated in an established hypertensive model of cardiac injury and fibrosis produced by infusion of angiotensin II/phenylephrine (AngII/PE). Mice were treated for 1 week to allow fibrosis development before injecting CD5/LNP-FAPCAR. Forty-eight hours after injection, a consistent population of FAPCAR-positive T cells (18–25%) were found in mice that received CD5/LNP-FAPCAR. Importantly, FAPCAR expression was transient in this setting, as no FAPCAR expression was found in splenic T cells 1 week after injection. Two weeks after LNP injection, cardiac function was analysed by echocardiography. A marked functional improvement was observed. Angiotensin II/phenylephrine-injured mice treated with CD5/LNP-FAPCAR exhibited normalized left ventricular (LV) end-diastolic and end-systolic volumes. Left ventricular diastolic function (E/e′) returned to uninjured levels. Left ventricular systolic function was also improved, as measured by ejection fraction and global longitudinal strain. A statistically significant improvement of the heart weight to body weight ratio (a measure of cardiac hypertrophy) was observed in treated animals. Histologic analysis showed a significant reduction of extracellular matrix (ECM) in injured mice treated with CD5/LNP-FAPCAR when compared with those treated with saline or IgG/LNP-FAPCAR controls, except for the persistence of perivascular fibrosis that resulted from activated fibroblasts that did not express FAP. No overt toxicity was observed, and LNP-mediated anti-FAPCAR expression was largely restricted to the spleen. Cardiac fibrosis plays a critical role in the development and progression of HF. Fibroblasts respond to heart injury and inflammation by chronically overproducing ECM that stiffens the heart, negatively affects cardiomyocyte health, and impairs systolic and diastolic function.2 However, antifibrotic treatments have demonstrated modest clinical benefits.3,4 An attractive novel strategy is to target activated cardiac fibroblasts with engineered T cells. This is possible because activated fibroblasts express unique cell surface markers that make them sufficiently different from quiescent, homeostatic fibroblasts and which can be specifically targeted.5 An early indication that this approach might be effective to reverse fibrosis and improve cardiac function came from mouse studies in the setting of pressure-overload injury, in which activated fibroblasts were genetically ablated.5 Chimeric antigen receptor T-cell technology is currently used for cancer treatment with remarkable success.6 In a previous study from the same group, using a mouse model of cardiac fibrosis and HF produced by the infusion of AngII/PE, FAPCAR T cells obtained ex vivo with a standard approach were shown to effectively restore cardiac function and mitigate the fibrotic response, even when the cells were delivered after fibrosis had already developed.7 Unlike the therapeutic scope in cancer, which requires eradication of the whole neoplastic tissue, CAR T cells in HF-associated fibrosis might produce a benefit even if only a fraction of activated fibroblasts are destroyed. Therefore, dosing might be titrated, and cells could be engineered to have transient activity, thus reducing potential long-term toxicity. This is of key importance since standard CAR T-cell therapeutic approaches would indeed be problematic in the management of human diseases associated with fibrosis, because activated fibroblasts have an important role in wound healing. Chimeric antigen receptor T cells that are reprogrammed with standard approaches to attack fibroblasts could survive in the body for months or even years, impairing wound healing over the longer term.7 Various mechanisms to produce transient CAR T cells have been described. The approach used here is based on mRNA to engineer CAR, so that the CAR is no longer expressed after the mRNA decays or after dilution due to cell division.8 When injected into mice, the LNP-encapsulated mRNA molecules were taken up by T cells and used as a template for the production of fibroblast-targeting CAR, effectively reprogramming the T cells to attack activated fibroblasts. The mRNAs are not integrated into T-cell DNA and survive a few days within T cells, that return to normal and do not target fibroblasts any longer. Despite this brief duration of activity, the reduction in cardiac fibroblasts led to an important reversal of interstitial fibrosis and substantially restored cardiac size and function.1 Moreover, standard CAR T-cell technology involves modifying patient’s T cells outside the body, which is technically challenging and expensive.6,7 The new approach could be used to avoid removing T cells from the patient by packaging modified mRNAs into LNPs capable of producing CAR T cells in vivo after injection. Although CAR T-cell therapy for cardiac diseases offers an exciting perspective, it is still a long way from translation into humans. Further studies in different experimental models of HF, possibly supported by cardiac magnetic resonance imaging,9 will be needed to optimize dosing strategy, LNP composition, and targeting approaches to enhance therapeutic effects and limit any potential toxicity, including potential off-target effects of FAPCAR T cells. It should be emphasized that, although interstitial fibrosis was reduced by FAPCAR treatment, perivascular fibrosis was not affected. Perivascular fibrosis and associated inflammation may play an important role in pathological cardiac remodelling by interfering with oxygen and nutrient supply to myocytes.2 Moreover, minor changes in the ECM can have a major impact on cardiac relaxation and the pumping properties of the heart.10 Thus, further studies should explore how CAR-T removal of fibroblasts affects the biophysical properties of cardiac scar tissue. Finally, in the current study, CAR T-cell therapy was started in the early stages of the disease process. Whether this approach can be used to treat more advanced stages of HF remains to be investigated. Conflict of interest: G.L. received grant support (to the Institution) for investigator-initiated research from American Heart Association, Italian National Health Service and Italian Minister of Education, University and Research. She is currently involved in the Research Programs of the Italian Cardiovascular Network. She received personal fees from Astra Zeneca, Boehringer Ingelheim, Novo Nordisk, and Daiichi Sankyo. C.P. received consultant and speaker fees from Acticor Biotech, Amgen, Bayer, Eli Lilly, GlaxoSmithKline, Tremeau, and Zambon, and grant support (to the Institution) for investigator-initiated research from AIFA (Italian Drug Agency), Bayer, Cancer Research UK, and European Commission; he chairs the Scientific Advisory Board of the International Aspirin Foundation.