CARdiac Immunotherapy: T Cells Engineered to Treat the Fibrotic Heart
Authors: Ronald J. Vagnozzi, Anne Katrine Z. Johansen, Jeffery D. Molkentin
Journal: Molecular Therapy (2019)

Progressive tissue fibrosis underlies numerous disease states, where it can diminish healthy organ function and regeneration after injury.1Wynn T.A. Cellular and molecular mechanisms of fibrosis.J. Pathol. 2008; 214: 199-210Crossref PubMed Scopus (3062) Google Scholar In the adult mammalian heart, excess fibrosis after myocardial infarction (MI) or in the setting of cardiomyopathy worsens cardiac function, leading to hypertrophy and, eventually, heart failure (HF),2Gourdie R.G. Dimmeler S. Kohl P. Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease.Nat. Rev. Drug Discov. 2016; 15: 620-638Crossref PubMed Scopus (199) Google Scholar While there are no current therapies that directly address cardiac fibrosis, a recent study3Aghajanian H. Kimura T. Rurik J.G. Hancock A.S. Leibowitz M.S. Li L. Scholler J. Monslow J. Lo A. Han W. et al.Targeting cardiac fibrosis with engineered T cells.Nature. 2019; 573: 430-433Crossref PubMed Scopus (246) Google Scholar has demonstrated a potential new strategy that employs chimeric antigen receptor (CAR) T cells engineered to specifically ablate activated fibroblasts (myofibroblasts), reducing fibrotic burden in a mouse model of cardiac injury. This proof-of-concept study in cardiac disease represents the first experimental assessment of CAR T cell immunotherapy outside of blood cancers. Here we discuss the therapeutic potential and additional questions to be addressed in moving forward with the concept of CAR T cells as an anti-fibrotic strategy. Fibrosis is the progressive accumulation and deposition of extracellular matrix (ECM) proteins, which are produced largely by tissue resident fibroblasts when converted to myofibroblasts. In the heart, the collagen-rich ECM serves as a structural scaffold for the contracting myocardium and as a robust signaling hub for secreted growth factors.4Bowers S.L. Banerjee I. Baudino T.A. The extracellular matrix: at the center of it all.J. Mol. Cell. Cardiol. 2010; 48: 474-482Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 5Hortells L. Johansen A.K.Z. Yutzey K.E. Cardiac Fibroblasts and the Extracellular Matrix in Regenerative and Nonregenerative Hearts.J. Cardiovasc. Dev. Dis. 2019; 6: 6Google Scholar Upon pathological stimuli, such as hemodynamic stress, myocardial ischemic damage, or persistent neurohumoral signaling, resident cardiac fibroblasts become activated and expand. These myofibroblasts then generate and secrete fibrillar and non-fibrillar collagens, matricellular proteins, and proteoglycans to generate a scar or interstitial fibrosis.2Gourdie R.G. Dimmeler S. Kohl P. Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease.Nat. Rev. Drug Discov. 2016; 15: 620-638Crossref PubMed Scopus (199) Google Scholar, 6Tallquist M.D. Molkentin J.D. Redefining the identity of cardiac fibroblasts.Nat. Rev. Cardiol. 2017; 14: 484-491Crossref PubMed Scopus (273) Google Scholar Despite extensive effort, there is a paucity of therapeutic options currently available to treat cardiac fibrosis clinically.2Gourdie R.G. Dimmeler S. Kohl P. Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease.Nat. Rev. Drug Discov. 2016; 15: 620-638Crossref PubMed Scopus (199) Google Scholar Hence, strategies to directly modulate or target the myofibroblasts to abate pathological cardiac fibrosis are desperately needed. CAR T cell technology has emerged within the past few years as part of the “immunotherapy revolution” for cancer treatment.7Sadelain M. Rivière I. Riddell S. Therapeutic T cell engineering.Nature. 2017; 545: 423-431Crossref PubMed Scopus (465) Google Scholar, 8June C.H. Sadelain M. Chimeric Antigen Receptor Therapy.N. Engl. J. Med. 2018; 379: 64-73Crossref PubMed Scopus (992) Google Scholar The overall approach with CAR T cells is to genetically engineer a hybrid T cell receptor consisting of (at minimum) an extracellular binding domain for a desired antigen, coupled to classical intracellular T cell activation and co-stimulation receptors (often CD3ζ and CD28, respectively7Sadelain M. Rivière I. Riddell S. Therapeutic T cell engineering.Nature. 2017; 545: 423-431Crossref PubMed Scopus (465) Google Scholar, 9Lim W.A. June C.H. The Principles of Engineering Immune Cells to Treat Cancer.Cell. 2017; 168: 724-740Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar) (Figure 1). T cells made to express this “chimeric” receptor recognize and initiate cytotoxic killing of cells expressing the target antigen. Adoptive transfer of CAR T cells targeting tumor antigens, notably the B cell antigen CD19, has shown considerable promise in patients with blood or bone marrow malignancies, including acute lymphoblastic and chronic lymphocytic leukemias and non-Hodgkin’s lymphoma.10Majzner R.G. Mackall C.L. Clinical lessons learned from the first leg of the CAR T cell journey.Nat. Med. 2019; 25: 1341-1355Crossref PubMed Scopus (223) Google Scholar Clinical trials are also underway to explore treatment of solid tumors with CAR T cell therapy, although the choice of surface antigen to mediate both selective and expansive malignant cell killing by CAR T cells remains problematic.7Sadelain M. Rivière I. Riddell S. Therapeutic T cell engineering.Nature. 2017; 545: 423-431Crossref PubMed Scopus (465) Google Scholar However, if a suitable antigen is identified that is uniformly expressed, such as demonstrated on the surface of cardiac myofibroblasts in the study by Aghajanian et al.,3Aghajanian H. Kimura T. Rurik J.G. Hancock A.S. Leibowitz M.S. Li L. Scholler J. Monslow J. Lo A. Han W. et al.Targeting cardiac fibrosis with engineered T cells.Nature. 2019; 573: 430-433Crossref PubMed Scopus (246) Google Scholar CAR T cell therapy could be applied. Aghajanian et al.3Aghajanian H. Kimura T. Rurik J.G. Hancock A.S. Leibowitz M.S. Li L. Scholler J. Monslow J. Lo A. Han W. et al.Targeting cardiac fibrosis with engineered T cells.Nature. 2019; 573: 430-433Crossref PubMed Scopus (246) Google Scholar first examined whether the basic principles of CAR T cell therapy could be co-opted to target myofibroblasts. They generated transgenic mice so that cardiac myofibroblasts could selectively express a xenogenic neo-antigen, ovalbumin peptide (OVA), via Cre-lox recombination. To achieve this, the authors used a mouse line with a tamoxifen-inducible Cre recombinase that is specifically expressed in myofibroblasts.11Kanisicak O. Khalil H. Ivey M.J. Karch J. Maliken B.D. Correll R.N. Brody M.J. J Lin S.C. Aronow B.J. Tallquist M.D. Molkentin J.D. Genetic lineage tracing defines myofibroblast origin and function in the injured heart.Nat. Commun. 2016; 7: 12260Crossref PubMed Scopus (442) Google Scholar As a model of cardiac injury, Aghajanian et al.3Aghajanian H. Kimura T. Rurik J.G. Hancock A.S. Leibowitz M.S. Li L. Scholler J. Monslow J. Lo A. Han W. et al.Targeting cardiac fibrosis with engineered T cells.Nature. 2019; 573: 430-433Crossref PubMed Scopus (246) Google Scholar infused mice for 4 weeks with a combination of angiotensin II (AngII) and phenylephrine (PE), a model that recapitulates the neurohumoral activation of fibroblasts seen in conditions such as chronic hypertension. The authors then created and adoptively transferred CAR T cells engineered to target OVA into these mice. They found that OVA-targeted CAR T cell transfer significantly diminished fibrotic burden as well as pathological cardiac hypertrophy. Importantly, these CAR T cells did not alter fibrosis or hypertrophy in control animals that were not induced to express the OVA antigen, indicating these effects were specific to ablation of myofibroblasts. Given these initial results, Aghajanian et al.3Aghajanian H. Kimura T. Rurik J.G. Hancock A.S. Leibowitz M.S. Li L. Scholler J. Monslow J. Lo A. Han W. et al.Targeting cardiac fibrosis with engineered T cells.Nature. 2019; 573: 430-433Crossref PubMed Scopus (246) Google Scholar then screened an extensive gene expression dataset from HF patients to identify potential endogenous antigens on cardiac myofibroblasts that could be suitable for therapeutic CAR T cell targeting. From this approach, the authors identified fibroblast activation protein (FAP), a membrane glycoprotein previously identified in cancer-associated12Garin-Chesa P. Old L.J. Rettig W.J. Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers.Proc. Natl. Acad. Sci. USA. 1990; 87: 7235-7239Crossref PubMed Scopus (492) Google Scholar and cardiac13Tillmanns J. Hoffmann D. Habbaba Y. Schmitto J.D. Sedding D. Fraccarollo D. Galuppo P. Bauersachs J. Fibroblast activation protein alpha expression identifies activated fibroblasts after myocardial infarction.J. Mol. Cell. Cardiol. 2015; 87: 194-203Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar fibroblasts, which they confirmed was induced in failing human hearts and the injured mouse heart. Importantly, FAP CAR T cells had already been successfully applied to ablate tumor-promoting cancer-associated stromal cells.14Wang L.C. Lo A. Scholler J. Sun J. Majumdar R.S. Kapoor V. Antzis M. Cotner C.E. Johnson L.A. Durham A.C. et al.Targeting fibroblast activation protein in tumor stroma with chimeric antigen receptor T cells can inhibit tumor growth and augment host immunity without severe toxicity.Cancer Immunol. Res. 2014; 2: 154-166Crossref PubMed Scopus (333) Google Scholar In the pivotal experiment, Aghajanian et al.3Aghajanian H. Kimura T. Rurik J.G. Hancock A.S. Leibowitz M.S. Li L. Scholler J. Monslow J. Lo A. Han W. et al.Targeting cardiac fibrosis with engineered T cells.Nature. 2019; 573: 430-433Crossref PubMed Scopus (246) Google Scholar showed that adoptively transferred FAP CAR T cells reached the heart, reduced cardiac fibrosis, and attenuated both hypertrophy and cardiac dysfunction in wild-type mice treated with AngII/PE (Figure 1). These data provided direct experimental evidence that CAR T cells engineered against an endogenous antigen on myofibroblasts could attenuate cardiac fibrosis. Taken together, the results of Aghajanian et al.3Aghajanian H. Kimura T. Rurik J.G. Hancock A.S. Leibowitz M.S. Li L. Scholler J. Monslow J. Lo A. Han W. et al.Targeting cardiac fibrosis with engineered T cells.Nature. 2019; 573: 430-433Crossref PubMed Scopus (246) Google Scholar represent the first step toward a potential new therapeutic paradigm of immunotherapy for the fibrotic heart. As with all groundbreaking paradigms that suggest a new therapeutic approach, key mechanistic studies to address unanswered questions are needed before clinical application should be attempted. For example, challenges and technical considerations of CAR T cell therapy in the context of cancer immunotherapy are well-documented in the literature15Sadelain M. Brentjens R. Rivière I. The promise and potential pitfalls of chimeric antigen receptors.Curr. Opin. Immunol. 2009; 21: 215-223Crossref PubMed Scopus (364) Google Scholar, 16Bonifant C.L. Jackson H.J. Brentjens R.J. Curran K.J. Toxicity and management in CAR T-cell therapy.Mol. Ther. Oncolytics. 2016; 3: 16011Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar and were appropriately acknowledged by Aghajanian et al.3Aghajanian H. Kimura T. Rurik J.G. Hancock A.S. Leibowitz M.S. Li L. Scholler J. Monslow J. Lo A. Han W. et al.Targeting cardiac fibrosis with engineered T cells.Nature. 2019; 573: 430-433Crossref PubMed Scopus (246) Google Scholar The choice of an antigen that is expressed uniquely or predominantly in the target cell population is crucial. In this case, FAP is also expressed in skin wounds,12Garin-Chesa P. Old L.J. Rettig W.J. Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers.Proc. Natl. Acad. Sci. USA. 1990; 87: 7235-7239Crossref PubMed Scopus (492) Google Scholar thus CAR T cell therapy against FAP might interfere with wound healing. However, Aghajanian et al.3Aghajanian H. Kimura T. Rurik J.G. Hancock A.S. Leibowitz M.S. Li L. Scholler J. Monslow J. Lo A. Han W. et al.Targeting cardiac fibrosis with engineered T cells.Nature. 2019; 573: 430-433Crossref PubMed Scopus (246) Google Scholar showed this did not occur in their model, at least over a classical 8-day time course of skin biopsy punch wounding in the mouse. Before moving forward, other potential cell types that might express FAP need to be excluded as well as more careful investigation of the role that FAP expressing cells play in acute tissue wound healing over time. The most frequently observed side effect of CAR T cell therapy is “cytokine release syndrome” (CRS), a systemic reactionary inflammatory response that can even produce effects akin to septic shock.16Bonifant C.L. Jackson H.J. Brentjens R.J. Curran K.J. Toxicity and management in CAR T-cell therapy.Mol. Ther. Oncolytics. 2016; 3: 16011Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar CRS is thought to be due to on-target killing of intended cells by CAR T cells and the subsequent inflammatory response it evokes. Another consideration is that patients with chronic HF, who would be the most likely candidates for FAP CAR T cell therapy, are often in a hyper-inflammatory state,17Mann D.L. Innate immunity and the failing heart: the cytokine hypothesis revisited.Circ. Res. 2015; 116: 1254-1268Crossref PubMed Scopus (426) Google Scholar and excess inflammation is both a predictor and driver of progressive cardiac dysfunction.18Lavine K.J. Mann D.L. Recognition of self-DNA drives cardiac inflammation: why broken hearts fail.Nat. Med. 2017; 23: 1400-1401Crossref PubMed Scopus (3) Google Scholar Recent data also suggest the innate immune response and particularly macrophages are mechanistic contributors to CRS following CAR T cell therapy.19Giavridis T. van der Stegen S.J.C. Eyquem J. Hamieh M. Piersigilli A. Sadelain M. CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade.Nat. Med. 2018; 24: 731-738Crossref PubMed Scopus (619) Google Scholar Of note, Aghajanian et al.3Aghajanian H. Kimura T. Rurik J.G. Hancock A.S. Leibowitz M.S. Li L. Scholler J. Monslow J. Lo A. Han W. et al.Targeting cardiac fibrosis with engineered T cells.Nature. 2019; 573: 430-433Crossref PubMed Scopus (246) Google Scholar observed a significant increase in macrophage infiltration into the heart and a modest elevation in serum levels of some pro-inflammatory cytokines with FAP CAR T cell therapy. In addition, recruitment and trafficking of T cell subsets are tightly controlled and subject to coordinated input from multiple signals that often reflect the overall inflammatory status of each patient.20Blanton R.M. Carrillo-Salinas F.J. Alcaide P. T-cell recruitment to the heart: friendly guests or unwelcome visitors?.Am. J. Physiol. Heart Circ. Physiol. 2019; 317: H124-H140Crossref PubMed Scopus (44) Google Scholar Thus, cardiac applications of CAR T cells will need to consider the overall immune profile of the subject and the phenotypic responses of both innate and adaptive immune cells to optimize potential efficacy as well as limit adverse reactions. Fortunately, such efforts are already emerging while using CAR T cells to treat leukemia and lymphoma in patients.21Linette G.P. Stadtmauer E.A. Maus M.V. Rapoport A.P. Levine B.L. Emery L. Litzky L. Bagg A. Carreno B.M. Cimino P.J. et al.Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma.Blood. 2013; 122: 863-871Crossref PubMed Scopus (764) Google Scholar, 22Burstein D.S. Maude S. Grupp S. Griffis H. Rossano J. Lin K. Cardiac Profile of Chimeric Antigen Receptor T Cell Therapy in Children: A Single-Institution Experience.Biol. Blood Marrow Transplant. 2018; 24: 1590-1595Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Another critical question is whether ablation of cardiac myofibroblasts is an appropriate therapeutic strategy for post-MI injury patients that might be at risk for having a subsequent MI. Myofibroblasts and their ability to acutely augment ECM production are critical in generating a fibrotic scar that heals the area of infarction injury to prevent ventricular wall rupture.2Gourdie R.G. Dimmeler S. Kohl P. Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease.Nat. Rev. Drug Discov. 2016; 15: 620-638Crossref PubMed Scopus (199) Google Scholar, 5Hortells L. Johansen A.K.Z. Yutzey K.E. Cardiac Fibroblasts and the Extracellular Matrix in Regenerative and Nonregenerative Hearts.J. Cardiovasc. Dev. Dis. 2019; 6: 6Google Scholar, 6Tallquist M.D. Molkentin J.D. Redefining the identity of cardiac fibroblasts.Nat. Rev. Cardiol. 2017; 14: 484-491Crossref PubMed Scopus (273) Google Scholar Thus, HF patients with persistent FAP CAR T cells used to control chronic fibrosis would be at greater risk for cardiac ventricular wall rupture if a subsequent MI injury event occurred. Even if the intended use was for acute and temporary ablation of cardiac myofibroblasts, infused CAR T cells can persist and function for many years.23Scholler J. Brady T.L. Binder-Scholl G. Hwang W.T. Plesa G. Hege K.M. Vogel A.N. Kalos M. Riley J.L. Deeks S.G. et al.Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells.Sci. Transl. Med. 2012; 4: 132ra53Crossref PubMed Scopus (463) Google Scholar To address these concerns, Aghajanian et al.3Aghajanian H. Kimura T. Rurik J.G. Hancock A.S. Leibowitz M.S. Li L. Scholler J. Monslow J. Lo A. Han W. et al.Targeting cardiac fibrosis with engineered T cells.Nature. 2019; 573: 430-433Crossref PubMed Scopus (246) Google Scholar proposed that FAP CAR T cells could be engineered in such a way to later eliminate them (kill switch gene) or multiple simultaneous antigens could be used to enhance selectivity.24Kloss C.C. Condomines M. Cartellieri M. Bachmann M. Sadelain M. Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells.Nat. Biotechnol. 2013; 31: 71-75Crossref PubMed Scopus (624) Google Scholar However, if the FAP CAR T cells were eliminated, progressive fibrosis in chronic HF would likely resume. In conclusion, the recent findings of Aghajanian et al.3Aghajanian H. Kimura T. Rurik J.G. Hancock A.S. Leibowitz M.S. Li L. Scholler J. Monslow J. Lo A. Han W. et al.Targeting cardiac fibrosis with engineered T cells.Nature. 2019; 573: 430-433Crossref PubMed Scopus (246) Google Scholar represent an innovative and promising conceptual advance in treating cardiac fibrosis. These results also support the potential use of CAR T cells for many indications beyond cancer therapy or even cardiovascular medicine, as any disease-associated cell type could be targeted for removal if selective surface antigens were identified. The “immunotherapy revolution” is truly at hand, bringing with it not only the potential for new treatments, but greater insight into the fundamental intersection of immunology with tissue homeostasis during aging and disease. R.J.V., A.K.Z.J., and J.D.M. drafted, reviewed, and edited the manuscript. J.D.M. was supported by grants from the NIH and by the Howard Hughes Medical Institute. R.J.V. was supported by a Career Development Award from the American Heart Association (19CDA34670044).