Use of a CAR-T cell targeting FAP in preparation of a drug for treating cardiac fibrosis in chronic stage of myocarditis

Abstract

The application provides an application of a FAP-targeted CAR-T cell in preparation of a drug for treating cardiac fibrosis in a chronic phase of myocarditis. It is found in the application that FAP-specific CAR-T cells can effectively target cells expressing mouse FAP proteins, and the CAR-T cells can effectively eliminate cells expressing mouse FAP proteins after being activated, the chimeric antigen receptor T cells effectively reduce cardiac fibrosis in a chronic phase of myocarditis in mice, and restore the function after inflammatory injury. The application develops a CAR-T therapy for myocardial fibrosis caused by a chronic phase of myocarditis, and the chimeric antigen receptor T cells expressing a targeted fibroblast activation protein can recognize activated myocardial fibroblasts, and significantly reduce cardiac fibrosis after inflammatory injury and restore the function.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to application of a CAR-T cell targeting FAP in preparation of a drug for treating cardiac fibrosis in chronic phase of myocarditis. BACKGROUND

[0002] Chronic inflammation in myocarditis patients can lead to myocardial fibrosis. After injury, activated fibroblasts secrete a large amount of fibrosis factors and extracellular matrix, which accumulate in the myocardial interstitium and perivascular space, and remodel the myocardium. Myocardial fibrosis is a remodeling process of myocardial interstitium, which is characterized by abnormal proliferation of myocardial interstitial fibroblasts, excessive accumulation of collagen fibers, significant increase in collagen concentration and volume fraction per unit mass of myocardium, and imbalance and abnormal distribution of various collagens. The reason is that myofibroblasts excessively deposit extracellular matrix proteins under chronic inflammatory response, leading to scar formation and tissue hardening, and gradually impairing heart function, eventually leading to heart failure. However, most of the drugs used in clinical practice to intervene myocarditis focus on anti-heart failure or arrhythmia, rather than fibrosis process, and cannot stop and eliminate activated fibroblasts.

[0003] A variety of stimuli can lead to the occurrence of myocardial fibrosis, including non-inflammatory pathways and inflammatory pathways, such as renin-angiotensin-aldosterone system pathway and TGF-β pathway. Myocardial injury caused by inflammation is one of the important reasons for myocardial fibrosis. In the process of chronic inflammation, a large number of immune cells, mainly monocytes and macrophages, gather and infiltrate, causing myocardial injury, and at the same time, secreting a large amount of pro-inflammatory mediators, including tumor necrosis factor-α, interleukin-6 and interleukin-1, forming a chronic inflammatory microenvironment. In this cell environment, a large number of cytokines and growth factors are released, including members of the transforming growth factor-β family and Wingless / Int-1, which are the main effector factors of the fibrosis process. TGF-β and Wnt1 bind to stem cell surface receptors and initiate downstream signaling, eventually leading to Smad2 / 3 and CBP / β-Catenin transcriptional regulators. This leads to up-regulation of target gene expression, further strengthening the differentiation of myofibroblasts and the production and secretion of collagen, laminin and fibronectin, thus promoting the process of myocardial fibrosis.

[0004] The current focus of clinical treatment of fibrosis is to reduce chronic inflammation, reduce the secretion of pro-inflammatory and pro-fibrotic factors, and inhibit the development of the renin-angiotensin-aldosterone system. This includes inhibiting TGF-β and SMAD through angiotensin receptor inhibitors or relaxin, regulating the balance between matrix metalloproteinases and tissue inhibitors of metalloproteinases, thereby treating fibrosis. However, activated myofibroblasts do not stop here, and the fibrosis process does not stop. Therefore, there is an urgent need to develop new treatment strategies to target fibrosis in the chronic phase of myocarditis.

[0005] Chimeric antigen receptor T cell therapy (CAR-T) has made breakthrough progress in the field of tumor, autoimmune and leukemia treatment. It extracts T cells from the immune system of patients, cultures and modifies T cells in vitro to make them have the ability to recognize and attack specific cancer cells, and then inject the modified T cells back into the patient's body. The immune response of T cells to cancer cells will eliminate cancer cells. The US Food and Drug Administration has approved CAR-T cell therapy for some forms of leukemia and lymphoma patients. Recent studies have shown that CAR-T cell therapy is also promising in solid tumors, autoimmune diseases, chronic infections, heart disease and diseases related to aging. Therefore, targeting non-cancer cells with CAR-T cells is a new attempt. The Jonathan-Epstein team has made a quantitative good effect in treating heart failure caused by fibrosis using CAR-T cell therapy, and studies have shown that CAR-T cell therapy can effectively inhibit the process of myocardial fibrosis in AngII-induced heart failure model mice. However, it is not yet clear whether CAR-T therapy can be applied to chronic inflammation-induced myocardial fibrosis.

[0006] Therefore, there is an urgent need to develop a CAR-T therapy for myocarditis-induced chronic myocardial fibrosis. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide the application of CAR-T cells targeting FAP in the preparation of a drug for treating myocarditis-induced chronic myocardial fibrosis. The present application finds that FAP is a potential candidate for targeting cardiac fibroblasts in myocarditis pathology, and develops a Fap-specific CAR-T cell (aFAP-CAR-T), which has excellent treatment effect on fibrosis in the chronic phase of myocarditis.

[0008] To achieve the purpose of the present application, the following technical solutions are adopted:

[0009] In a first aspect, the present application provides the use of a substance targeting Fap gene and / or FAP protein in the preparation of a drug for treating myocarditis-induced chronic myocardial fibrosis.

[0010] Preferably, the substance targeting Fap gene as a target is an inhibitor that reduces the activity and / or expression of Fap gene.

[0011] Preferably, the substance targeting FAP protein as a target is an inhibitor that reduces the expression and function of FAP protein, or an antibody targeting FAP protein, a CAR molecule targeting FAP protein, or a CAR cell targeting FAP protein.

[0012] Preferably, the inhibitor comprises: any one or a combination of two of a small molecule compound, an interfering RNA vector expressing a Fap gene, or a vector of a Fap gene knockdown or knock-out system.

[0013] Preferably, the vector comprises: any one or a combination of at least two of an adenovirus, an adeno-associated virus, a lentivirus, a plasmid, a liposome, or a nanoparticle.

[0014] Preferably, the CAR cell targeting the FAP protein comprises: any one of a CAR-T, a CAR-NK, a CAR-NKT, a CAR-M (CAR-macrophage), a CAR Treg, or a CAR-gammadelta T.

[0015] In the present application, it is found that FAP-specific CAR-T cells can effectively target cells expressing mouse FAP protein, and the CAR-T cells can effectively eliminate cells expressing mouse FAP protein after being activated.

[0016] The present application also provides a use of the CAR-T cell targeting the FAP protein in the preparation of a drug for treating cardiac fibrosis in the chronic phase of myocarditis.

[0017] The present application develops a CAR-T therapy for myocardial fibrosis caused by the chronic phase of myocarditis. Through RNA sequencing data of myocarditis patients, dilated cardiomyopathy patients and healthy people, specific genes of myocarditis fibrosis in the chronic phase and endogenous targets of cardiac fibrosis are determined. A CAR-T is constructed to express a chimeric antigen receptor targeting the target. Subsequently, an experimental autoimmune myocarditis (EAM) model and a viral myocarditis model (CVB9) are constructed to simulate chronic myocardial fibrosis associated with inflammation. The CAR-T is injected into the myocarditis mice through the vein regularly to study its effect on treating myocardial fibrosis in the chronic phase of myocarditis. According to the research results of EAM, CVB9 mouse myocardial samples and human myocarditis patient samples, fibroblast activation protein (FAP) is the fibrosis gene with the highest expression in patients in the chronic phase of myocarditis, and its expression is related to the degree of cardiac fibrosis. Therefore, FAP is selected as a marker of myocarditis fibrosis in the chronic phase. Chimeric antigen receptor T cells (aFAP-CAR-T) targeting fibroblast activation protein can recognize activated myocardial fibroblasts and significantly reduce cardiac fibrosis and restore function after inflammatory injury in mice. This method effectively reduces cardiac fibrosis in the chronic phase of myocarditis in mice and restores function after inflammatory injury. These findings provide evidence for the development of CAR-T in the treatment of immune-related diseases of the heart.

[0018] Preferably, the CAR molecule targeting the FAP protein specifically binds to the FAP protein expressed by cardiac fibroblasts, and the amino acid sequence of the extracellular recognition domain of the CAR molecule is selected from:

[0019] an amino acid sequence as shown in SEQ ID NO. 2; or an amino acid sequence formed by substitution, addition or deletion of amino acids of the amino acid sequence as shown in SEQ ID NO. 2, which has the function of binding to FAP.

[0020] Preferably, the CAR molecule comprises, in sequence, a signal peptide, an extracellular recognition domain, a transmembrane domain and a signaling domain.

[0021] Preferably, the transmembrane domain is a CD28 hinge region and a CD28 transmembrane region.

[0022] Preferably, the signaling domain is a CD28 intracellular signaling domain and a CD3z intracellular signaling domain.

[0023] Preferably, there is a FLAG tag between the signal peptide and the extracellular recognition domain.

[0024] Preferably, the CAR molecule comprises an amino acid sequence as shown in SEQ ID NO. 10.

[0025] Preferably, the CAR-T cell is prepared by a preparation method comprising the following steps:

[0026] constructing an expression vector containing a nucleic acid molecule encoding a CAR molecule targeting FAP;

[0027] co-transfecting the expression vector and a packaging helper plasmid into a mammalian cell to obtain a recombinant lentivirus;

[0028] transfecting the recombinant lentivirus into T cells for expression to obtain a CAR-T cell, wherein the cell expresses a CAR molecule targeting FAP.

[0029] Preferably, the expression vector comprises a viral vector.

[0030] Preferably, the viral vector comprises any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector, preferably a retroviral vector.

[0031] Preferably, the nucleic acid molecule encoding a CAR molecule targeting FAP comprises SEQ ID NO. 9.

[0032] Preferably, the amino acid sequence of the CAR molecule targeting FAP comprises SEQ ID NO. 10.

[0033] In a second aspect, the present application provides a chimeric antigen receptor targeting FAP, wherein the chimeric antigen receptor specifically binds to FAP protein expressed by cardiac fibroblasts, and an amino acid sequence of an extracellular recognition domain of the chimeric antigen receptor is selected from:

[0034] an amino acid sequence as shown in SEQ ID NO. 2; or an amino acid sequence formed by substitution, addition or deletion of amino acids of the amino acid sequence as shown in SEQ ID NO. 2, having a function of binding to FAP.

[0035] In the present application, the amino acid sequence formed by substitution, addition or deletion of amino acids of the amino acid sequence as shown in SEQ ID NO. 2 has at least 90% identity to the amino acid sequence as shown in SEQ ID NO. 2, preferably at least 95% identity.

[0036] Preferably, the chimeric antigen receptor comprises a signal peptide, an extracellular recognition domain, a transmembrane domain and a signaling domain in series.

[0037] Preferably, the transmembrane domain is a CD28 hinge region and a CD28 transmembrane region.

[0038] Preferably, the signaling domain is a CD28 intracellular signaling domain and a CD3z intracellular signaling domain.

[0039] Preferably, the chimeric antigen receptor comprises an amino acid sequence as shown in SEQ ID NO. 10.

[0040] In a third aspect, the present application provides a nucleic acid molecule encoding the FAP-targeting chimeric antigen receptor of the second aspect.

[0041] In a fourth aspect, the present application provides an expression vector comprising at least one copy of the nucleic acid molecule of the third aspect.

[0042] Preferably, the expression vector comprises a viral vector.

[0043] Preferably, the viral vector comprises any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector, preferably a retroviral vector.

[0044] In a fifth aspect, the present application provides a recombinant lentivirus prepared by a preparation method comprising the following step: co-transfecting the viral vector of the fourth aspect and a packaging helper plasmid into a mammalian cell to obtain the recombinant lentivirus.

[0045] In a sixth aspect, the present application provides a FAP-targeting chimeric antigen receptor T cell prepared by a preparation method comprising the following step: transfecting the recombinant lentivirus of the fifth aspect into a T cell to express, to obtain the chimeric antigen receptor T cell, wherein the cell expresses the FAP-targeting chimeric antigen receptor of the second aspect.

[0046] In the present application, the preparation method of the FAP-targeted chimeric antigen receptor T cells comprises the following steps:

[0047] (1) constructing an expression vector encoding a FAP-targeted chimeric antigen receptor;

[0048] (2) co-transfecting the expression vector in step (1) and a packaging plasmid into a virus packaging cell to prepare a recombinant lentivirus;

[0049] (3) introducing the recombinant lentivirus in step (2) into T cells to prepare FAP-targeted chimeric antigen receptor T cells.

[0050] In a seventh aspect, the present application provides a pharmaceutical composition comprising the FAP-targeted chimeric antigen receptor T cells of the sixth aspect.

[0051] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0052] In an eighth aspect, the present application provides use of the FAP-targeted chimeric antigen receptor T cells of the sixth aspect and / or the pharmaceutical composition of the seventh aspect in the preparation of a drug for treating cardiac fibrosis in the chronic phase of myocarditis.

[0053] Compared with the prior art, the present application has the following beneficial effects:

[0054] (1) The present application finds that the expression of FAP is positively correlated with the degree of fibrosis, and FAP is a potential candidate for targeting cardiac fibroblasts in the pathology of myocarditis.

[0055] (2) The FAP-specific CAR T cells (same as "CAR-T cells") constructed in the present application can effectively target and lyse cells expressing mouse FAP protein, and the alphaFAP-CAR T has excellent therapeutic effect on fibrosis in the chronic phase of myocarditis. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 is a single-cell sequencing analysis diagram of human myocardial samples.

[0057] Figure 2 is an immunohistochemical staining diagram of human myocardial samples.

[0058] Figure 3 is a schematic diagram of construction of mouse Fap-specific CAR T cells.

[0059] Figure 4 is a verification diagram of mouse Fap-specific CAR T cells.

[0060] Figure 5Figure of experimental results of αFAP-CAR T cell targeted therapy for cardiac fibrosis in EAM model mice.

[0061] Figure 6 Figure of experimental results of αFAP-CAR T cell targeted therapy for cardiac fibrosis in CVB3 model mice.

[0062] Figure 7 Figure of results of in vivo biological safety evaluation. DETAILED DESCRIPTION

[0063] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application, and should not be regarded as specific limitations on the present application.

[0064] Unless specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in the art or according to the product instructions are used. Unless the manufacturer of the reagent or instrument is specified, it is a conventional product that can be commercially available through a regular channel.

[0065] Example 1

[0066] 1. Analysis of human heart expression data

[0067] Human heart samples were obtained from patients undergoing heart transplantation surgery (Table 1). All human hearts collected were protected with in situ cardioplegia. Echocardiography was performed before tissue collection to assess real-time structure and function in vivo. The clinical study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the Review Committee of Fu Wai Hospital. All participants provided written informed consent before participating in the study. Tissue specimens were frozen with liquid nitrogen before use and stored at a temperature of -80°C.

[0068] Table 1

[0069]

[0070]

[0071] Variables are expressed as frequency (percentage), mean ± SD. Bold indicates statistical significance. HTx, heart transplantation. MACE: major adverse cardiovascular events. ICD, implantable cardioverter-defibrillator. CRT, cardiac resynchronization therapy. CRT-D, cardiac resynchronization therapy defibrillator. NYHA: New York Heart Association. RV: right ventricle. LV, left ventricle. RVEDD: right ventricular end-diastolic diameter. LVEDD, left ventricular end-diastolic diameter. LVEF, left ventricular ejection fraction.

[0072] Total RNA was extracted from human heart tissue samples using the miRNeasy Kit (Qiagen) including DNase treatment. For RNA sequence analysis, library preparation was performed using the Illumina truSeq stranded mRNA kit followed by the Nugen Ovation amplification kit. Resulting FASTQ files were assessed for quality control using the FastQC program. FASTQ files were aligned to the human reference genome (hGRC37 / Hg19) using the STAR aligner46. Duplicate reads were marked using the MarkDuplicates program of the Picard tools. R packages were used to calculate read counts per gene annotated to Ensembl (v.75) genes and remove duplicate reads. Gene counts expressed in counts per million (CPM) were first normalized using the TMM method in the edgeR R package, followed by removal of genes with CPM <1 in 25% of samples and considered as low expressed genes. Data were transformed using the VOOM function of the limma R package. Differential gene expression was analyzed using a linear model of the limma package.

[0073] 2. Construction of mouse autoimmune myocarditis model and viral myocarditis model

[0074] All animal experiments involved in the present application were approved by the Animal Ethics and Welfare Committee of Shenzhen Hospital, Fu Wai Hospital, Chinese Academy of Medical Sciences. Six-week-old male Balb / c mice were purchased from Guangzhou Jisui Yaoke Co., Ltd. Mice were acclimated for at least one week before the experiment and were provided with fresh food and water. The EAM mouse model was established by subcutaneous injection of 250 μg of α-MyHC peptide (Ac-RSLKLMATLFSTYASADR-OH; AnaSpec, AS-62554), which was emulsified with complete Freund's adjuvant (Sigma, F5881, 1:1, weight ratio) on day 0 and day 7. The EAM mouse model was successfully established 14 days after injection of α-MyHC. The method for modeling viral myocarditis in mice: select 6-week-old male inbred Balb / c mice, weighing (15-19) g, and divide them into an experimental group and a normal group. After the injection site was routinely disinfected, 10 5 i.e. 100 TCID 50 / 0.1 mL of CVB30.1 mL, and the normal group was injected intraperitoneally with an equal amount of 0.1 mmol / L phosphate buffer 0.1 mL. The CVB3 mouse model was successfully established on day 7 when the virus was inoculated.

[0075] 3. Plasmid construction

[0076] DNA sequences encoding mouse codon-optimized anti-FAP scFv (clone 73.3 [PMID: 24778279]), mouse CD28 hinge, transmembrane and signaling endodomain, mouse CD3z endodomain were cloned into MSCV retroviral constructs. A FLAG tag was introduced at the 5' end of the 73.3 scFv (downstream of the signal peptide). A vector expressing mouse Fap gene was constructed by cloning the mFap cDNA into the gamma retroviral vector SFG, and introducing a double helix IRES-DNGFR marker upstream of it [PMID: 32015548].

[0077] The nucleotide sequence encoding the mouse codon-optimized anti-FAP scFv is: SEQ ID NO. 1:

[0078] CAGGTGCAGCTGAAAGAGTCCGGCGGAGGACTGGTGCAGCCTGGCGGATCTCTGAAGCTGAGCTGTGCTGCCAGCGGCTTCACCTTCAGCAGCTACGGCATGAGCTGGGTGCGACAGACCGCCGACAAGAGACTGGAACTGGTGGCTACCACCAACAACAACGGCGGCGTGACCTACTACCCCGACAGCGTGAAGGGCAGATTCACCATCTCCAGAGACAACGCCAAGAACACCCTGTACCTGCAGATGAGCAGCCTGCAGAGCGAGGACACCGCCATGTACTACTGCGCCAGATACGGCTACTACGCCATGGATTACTGGGGCCAGGGCATCAGCGTGACCGTGTCTAGCGGAGGCGGCGGATCTGGCGGAGGGGGATCTAGTGGCGGAGGCTCTGACGTGCTGATGACCCAGACACCTCTGAGCCTGCCAGTGTCCCTGGGCGACCAGGCCAGCATCAGCTGTAGAAGCAGCCAGAGCATCGTGCACAGCAACGGCAACACCTACCTGGAATGGTATCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTGTCCAACAGATTCAGCGGCGTGCCCGACAGATTCTCCGGCAGCGGCTCTGGCACCGACTTCACCGTGAAGATCTCCAGGGTGGAAGCCGAGGACCTGGGCGTGTACTACTGTTTTCAAGGCAGCCACGTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG.

[0079] The amino acid sequence of the anti-mouse FAP scFv is: SEQ ID NO. 2:

[0080] QVQLKESGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTADKRLELVATTNNNGGVTYYPDSVKGRFTISRDNAKNTLYLQMSSLQSEDTAMYYCARYGYYAMDYWGQGISVTVSSGGGGSGGGGSSGGGSDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTVKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK.

[0081] The nucleotide sequence encoding the mouse CD28 hinge, transmembrane and signaling endodomain is: SEQ ID NO. 3:

[0082] ATTGAGTTCATGTACCCTCCGCCTTACCTAGACAACGAGAGGAGCAATGGAACTATTATTCACATAAAAGAGAAACATCTTTGTCATACTCAGTCATCTCCTAAGCTGTTTTGGGCACTGGTCGTGGTTGCTGGAGTCCTGTTTTGTTATGGCTTGCTAGTGACAGTGGCTCTTTGTGTTATCTGGACAAATAGTAGAAGGAACAGACTCCTTCAAAGTGACTACATGAACATGACTCCCCGGAGGCCTGGGCTCACTCGAAAGCCTTACCAGCCCTACGCCCCTGCCAGAGACTTTGCAGCGTACCGCCCC.

[0083] The amino acid sequence of the mouse CD28 hinge, transmembrane and signaling endodomain is: SEQ ID NO. 4:

[0084] IEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFCYGLLVTVALC VIWTNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRP

[0085] The nucleotide sequence encoding the mouse CD3z endodomain is: SEQ ID NO. 5:

[0086] AGAGCAAAATTCAGCAGGAGTGCAGAGACTGCTGCCAACCTGCAGGACCCCAACCAGCTCTACAATGAGCTCAATCTAGGGCGAAGAGAGGAATATGACGTCTTGGAGAAGAAGCGGGCTCGGGATCCAGAGATGGGAGGCAAACAGCAGAGGAGGAGGAACCCCCAGGAAGGCGTATACAATGCACTGCAGAAAGACAAGATGGCAGAAGCCTACAGTGAGATCGGCACAAAAGGCGAGAGGCGGAGAGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGCACTGCCACCAAGGACACCTATGATGCCCTGCATATGCAGACCCTGGCCCCTCGC.

[0087] The amino acid sequence of the mouse CD3z endodomain is: SEQ ID NO. 6:

[0088] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQ EGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR.

[0089] The nucleotide sequence encoding the signal peptide and the FLAG tag is: SEQ ID NO. 7:

[0090] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACTACAAAGACGATGACGACAAG.

[0091] The amino acid sequence of the signal peptide and the FLAG tag is: SEQ ID NO. 8:

[0092] MALPVTALLLPLALLLHAARPDYKDDDDK.

[0093] The nucleotide sequence of the chimeric antigen receptor targeting mouse FAP is: SEQ ID NO. 9:

[0094]

[0095] Amino acid sequence of the chimeric antigen receptor targeting mouse FAP: SEQ ID NO. 10:

[0096] MALPVTALLLPLALLLHAARPDYKDDDDKQVQLKESGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTADKRLELVATTNNNGGVTYYPDSVKGRFTISRDNAKNTLYLQMSSLQSEDTAMYYCARYGYYAMDYWGQGISVTVSSGGGGSGGGGSSGGGSDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTVKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKTRIEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRPRAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR.

[0097] 4. Retrovirus production

[0098] Retroviruses encoding CAR molecules were produced following a previously reported protocol [PMID: 32015548]. Briefly, transport vector and ecopack vector were mixed in a 3:1 ratio and then used to transfect HEK293T cells using Transfection reagent (Mirus) was used to transfect HEK293T cells following the manufacturer’s instructions. Supernatants containing retroviruses were collected 48 and 72 hours after transfection, filtered, snap-frozen and stored at -80°C until use.

[0099] 5. Mouse CAR T cell production

[0100] On day 0, mouse CAR T cells were generated as previously reported [PMID:32015548]. Briefly, splenocytes were harvested from 6-8 weeks old Balb / c mice and T cells were sorted using the MojoSort Mouse CD3 T Cell Isolation Kit (Biolegend). Sorted T cells were cultured in mouse T cell medium (RPMI 1640 + 10% FBS + 2mM Glutamax + 100mM b-mercaptoethanol + 1X NEAA + 1mM sodium pyruvate) and stimulated for two days with plate-bound anti-mouse CD3 / CD28 antibodies (eBioscience). Activated mouse T lymphocytes were transduced with retroviral supernatant using retronectin-coated plates [PMID:32015548]. Two days after transduction (day 4), T cells were collected and expanded in T cell medium containing rhIL-7 (10 ng / ml) and rhIL-15 (5 ng / ml). All functional tests and in vivo experiments were performed on day 5.

[0101] 6. Generation of human lymphoma cell line expressing mouse FAP

[0102] Retroviruses encoding mouse Fap were prepared using a two-way packaging vector (RDF114) and human lymphoma cell line (Daudi) was transduced using spinoculation. Expression of mFap was detected according to staining of the surface of mouse FAP by DNGFR and anti-mouse FAP monoclonal antibody (clone 73.3, Millipore).

[0103] 7. Flow cytometry analysis

[0104] For cell surface staining, cells were incubated with antibodies for 15 min at room temperature or for 30 min at 4°C. The following antibodies for flow cytometry analysis were from Biolegend: APC-conjugated anti-human CD20 (clone 2H7), BV421-conjugated anti-FLAG (clone L5), FITC-conjugated anti-mouse CD69 (clone H1.2F3), Alexa-Fluor 700-conjugated anti-mouse CD8 (clone 53-6.7), PE-Cy5-conjugated anti-mouse CD4 (clone Gk1.5), PE-conjugated anti-human NGFR (clone ME20.4), Alexa-Fluor 647-conjugated goat anti-mouse IgG (clone Poly4053); the following antibody was from BD Bioscience: PE-conjugated anti-mouse CD3 (clone 500A2). Flow cytometry data were acquired on a Novocyte Quanteon (Agilent) using NovoExpress software and flow data were analyzed using FlowJo software (v10.6.2, Tree Star).

[0105] 8. Histological examination

[0106] Heart tissue from mouse and human samples were collected, fixed in 4% (v / v) formalin solution overnight, embedded in paraffin, and cut into 5 pm-thick sections. The sections were then stained with H&E, immunohistochemistry, and immunofluorescence for histopathological evaluation. H&E and immunohistochemistry images were examined by a digital microscope. Fluorescence images were observed under a stereomicroscope (Leica DMi8, Leica Microsystems, Germany) and quantified by ImageJ.

[0107] 9. Statistical analysis

[0108] Continuous variables were expressed as mean ± SD and compared using the Kruskal-Wallis rank test. Categorical data were expressed as counts and percentages and compared using the χ2test. Spearman’s correlation coefficient was used to test the correlation between continuous variables. Survival curves were generated using the Kaplan-Meier method and compared by the log-rank test. Univariate and multivariate Cox proportional hazards analysis were used to test the association between time to outcome event and baseline covariates. All analyses were performed using Graphpad Prism version 9.0 software (GraphPad Software Corp, Boston, USA).

[0109] 10. Results

[0110] First, this example identified endogenous proteins expressed by activated cardiac fibroblasts resulting from myocarditis that can be designed (or transgenically) into T cells as antigen-specific targets. RNA sequencing was performed on clinical myocardial tissue samples from 19 heart transplant patients (4 myocarditis patients, 15 DCM patients, and 15 normal patients, Table 1), and differential gene expression analysis was performed on the results. The two groups of patients were similar in clinical presentation, including gender, age at symptoms / heart transplant, and arrhythmias. We found that serum NT-proBNP levels were higher in the DCM group than in the myocarditis group (3823.3 ± 1734.9 v.s. 1622.2 ± 264.0 fmol / ml, P < 0.001). In addition, the myocarditis group had a thicker interventricular septum but lower LVEDD and LVEF.

[0111] Figure 1Figure 2. Single-cell RNA sequencing analysis of human myocardial samples. (A) Heatmap of gene expression changes (fold change) of DCM and MYO patient heart fibroblasts compared to non-failing hearts, n = 15 DCM hearts, 4 MYO hearts. (B) Umap plot of heart fibroblast clusters in heart failure and normal conditions, data from previous study (GSE145154). (C) Violin plot of FAP expression in fibroblast clusters.

[0112] Figure 2 Figure 3. Immunohistochemical staining of human myocardial samples. (D) H&E, Masson’s trichrome fibrosis staining (blue), Sirius red fibrosis staining (red), immunohistochemical FAP (green) expression and DAPI (blue) staining of nine non-failing heart and MYO human left ventricular free wall sections. Representative images of two independent experiments show similar results. (E-F) Quantification of ventricular fibrosis and FAP expression. (G) Correlation of fibrosis percentage and FAP expression.

[0113] Results showed that several fibroblast-specific genes 27 were upregulated in myocardium of DCM or myocarditis patients compared to control, non-failing donor hearts (Figure 2A). Among these genes, FAP expression and upregulation fold change were most prominent. FAP is a cell surface glycoprotein that has been previously demonstrated to be expressed during embryonic development at sites of active tissue remodeling (e.g. during wound repair), during tissue fibrosis processes and in various tumors, but only at low levels in most normal adult tissues in mice and humans. FAP expression has been previously observed in human heart after acute myocardial infarction injury26, 28. According to previous study on human heart (including normal condition) fibroblast profiling (GEO: GSE145154)29, we found that FAP was only expressed in a specific fibroblast subset named myofibroblast (FB_7) (Figure 2B, C). We further found that in failing myocarditis and DCM human left ventricular tissues, heart fibroblasts (but not cardiomyocytes) strongly expressed FAP, while FAP expression was minimal in normal human heart (Figure 2D, E and F). Moreover, FAP expression positively correlated with fibrosis degree (Figure 2G). Therefore, FAP is a potential candidate to target heart fibroblasts in myocarditis pathology. Figure 1 Figure 1 Figure 2 Figure 2

[0114] ​​​​We hypothesized that targeted elimination of FAP-positive cells could improve fibrotic symptoms during myocarditis. Therefore, we constructed a mouse FAP-specific CAR construct containing the clonal 73.3scFv sequence, mouse CD28 and CD3 intracellular domains, and a FLAG tag as a detection marker. We expressed this CAR molecule in primary mouse T cells using retroviral transduction.

[0115] Figure 3 This diagram illustrates the construction of mouse Fap-specific CAR T cells; (A) shows the construction and validation of mouse Fap-specific CAR T cells (n=3). (B) shows the overexpression of the mouse Fap gene and FACS sorting of mFap-positive target cells. (C) shows a schematic diagram of the functional experiment validating the specificity of mouse Fap-specific CAR T cells.

[0116] Figure 4 This is a validation diagram of mouse Fap-specific CAR T cells; where (D) shows CD4+ cells co-cultured with mFap-positive or mFap-negative target cells. + and CD8 + Expression of CD69 activation markers on T cells, n=3 (Ctrl), n=4 (αFAP-CAR Ts). (E) Percentage of mFAP-positive or negative targets lysed when co-cultured with Ctrl or αFAP-CAR T cells, n=3 (Ctrl), n=4 (αFAP-CAR Ts). (F) Cytokines produced by Ctrl or αFAP-CAR T cells when co-cultured with mFAP-positive or negative targets.

[0117] The results showed that CAR molecules were expressed efficiently. Figure 3 To verify the function of the expressed CAR molecule, we designed a human cell line that ectopically expresses the mouse Fap (mFap) gene and sorted Fap-positive cells on the surface using FACS. Figure 3 Using this mFap-positive cell line and a wild-type cell line lacking mFap expression, we tested the specificity and functional targeting of CAR T cells. Figure 3 In the study, Fap-specific CAR T cells were activated by mFap-positive targets, with untransduced T cells serving as a control (Ctrl). We found that, based on CD69 expression, Fap-specific CAR T cells could be definitively activated by mFap-positive targets. In contrast, Ctrl T cells remained unactivated. Figure 4 (Middle D). Furthermore, activation of Fap-specific CAR T cells can effectively clear mFAP-positive target cells. However, there was no mFAP-negative control ( Figure 4Figure 6. Fap-specific CAR T cells produce large amounts of cytokines (TNFa, IFNg and IL-2) upon co-culture with mFap-positive target cells Figure 4 Figure 6. Fap-specific CAR T cells produce large amounts of cytokines (TNFa, IFNg and IL-2) upon co-culture with mFap-positive target cells

[0118] Figure 5 Figure 7. Experimental results of aFAP-CAR T cell targeted therapy for cardiac fibrosis in EAM model mice. (A) Schematic diagram of aFAP-CAR T cell targeting cardiac fibroblasts. (B) M-mode echocardiography and digital photographs of mice treated with saline (top), EAM (middle) or aFAP-CAR T cells (bottom). (C) H&E staining, fibrosis Masson's trichrome staining (blue) and FAP (red), CD68 (red), LY6C (green), MPO (yellow), CD3 (red), CD4 (green) and CD8 (yellow) immunofluorescence staining of coronary artery sections of EAM mice hearts after 4 weeks of saline or aFAP-CAR T cell treatment. (D, E) Statistics of body weight and ejection fraction of each group. (F, G) Quantification of ventricular fibrosis and FAP expression. (H, I and J) Staining of CD68 + , LY6C + , MPO + cells in heart sections after different treatments. (K, L and M) Staining of CD3 + , CD4 + and CD8 + T cells in heart sections after different treatments. Representative images of two independent experiments with similar results. Scale bar, 100 pm. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; NS, not significant; one-way ANOVA between groups.

[0119] To investigate the feasibility of targeting FAP as an endogenous cardiac fibroblast target for immunotherapy, we constructed an autoimmune myocarditis (EAM) mouse model and a viral myocarditis model. Cardiac fibrosis was very severe in Balb / c mice six weeks after Myhc sensitization. The presence of FAP in activated cardiac fibroblasts has been confirmed in this EAM model.

[0120] Immunohistochemistry showed that FAP was not detected in the control group heart, but was significantly expressed in the activated fibroblast tissue sample of the EAM mouse six weeks later Figure 6Figure 6. Anti-aFAP-CAR T cells significantly reduced the degree of fibrosis in EAM mice. (A) To eliminate the heart fibroblasts expressing FAP in the EAM chronic phase model, we injected anti-aFAP-CAR T cells immediately after the EAM model was established (day 14) and injected again after 2 weeks (day 28) Figure 6 Figure 6. Anti-aFAP-CAR T cells significantly reduced the degree of fibrosis in EAM mice. (A) To eliminate the heart fibroblasts expressing FAP in the EAM chronic phase model, we injected anti-aFAP-CAR T cells immediately after the EAM model was established (day 14) and injected again after 2 weeks (day 28) Figure 6 Figure 6. Anti-aFAP-CAR T cells significantly reduced the degree of fibrosis in EAM mice. (A) To eliminate the heart fibroblasts expressing FAP in the EAM chronic phase model, we injected anti-aFAP-CAR T cells immediately after the EAM model was established (day 14) and injected again after 2 weeks (day 28) Figure 6 Figure 6. Anti-aFAP-CAR T cells significantly reduced the degree of fibrosis in EAM mice. (A) To eliminate the heart fibroblasts expressing FAP in the EAM chronic phase model, we injected anti-aFAP-CAR T cells immediately after the EAM model was established (day 14) and injected again after 2 weeks (day 28) Figure 6 Figure 6. Anti-aFAP-CAR T cells significantly reduced the degree of fibrosis in EAM mice. (A) To eliminate the heart fibroblasts expressing FAP in the EAM chronic phase model, we injected anti-aFAP-CAR T cells immediately after the EAM model was established (day 14) and injected again after 2 weeks (day 28) Figure 6 Figure 6. Anti-aFAP-CAR T cells significantly reduced the degree of fibrosis in EAM mice. (A) To eliminate the heart fibroblasts expressing FAP in the EAM chronic phase model, we injected anti-aFAP-CAR T cells immediately after the EAM model was established (day 14) and injected again after 2 weeks (day 28) + Figure 6. Anti-aFAP-CAR T cells significantly reduced the degree of fibrosis in EAM mice. (A) To eliminate the heart fibroblasts expressing FAP in the EAM chronic phase model, we injected anti-aFAP-CAR T cells immediately after the EAM model was established (day 14) and injected again after 2 weeks (day 28) + Figure 6. Anti-aFAP-CAR T cells significantly reduced the degree of fibrosis in EAM mice. (A) To eliminate the heart fibroblasts expressing FAP in the EAM chronic phase model, we injected anti-aFAP-CAR T cells immediately after the EAM model was established (day 14) and injected again after 2 weeks (day 28) + Figure 6. Anti-aFAP-CAR T cells significantly reduced the degree of fibrosis in EAM mice. (A) To eliminate the heart fibroblasts expressing FAP in the EAM chronic phase model, we injected anti-aFAP-CAR T cells immediately after the EAM model was established (day 14) and injected again after 2 weeks (day 28) Figure 6 Figure 6. Anti-aFAP-CAR T cells significantly reduced the degree of fibrosis in EAM mice. (A) To eliminate the heart fibroblasts expressing FAP in the EAM chronic phase model, we injected anti-aFAP-CAR T cells immediately after the EAM model was established (day 14) and injected again after 2 weeks (day 28)

[0121] Figure 6Figure 6. Experimental results of aFAP-CAR T cell targeted therapy for CVB3 model mice cardiac fibrosis, in which (A) experimental schematic of aFAP-CAR T cell targeting cardiac fibroblasts. (B) M-mode echocardiography and digital photographs of mice treated with saline (top), CVB3 (middle), or aFAP-CAR T cells (bottom). (C) H&E staining, fibrosis Masson's trichrome staining (blue), and FAP (red), CD68 (red), LY6C (green), MPO (yellow), CD3 (red), CD4 (green), and CD8 (yellow) immunofluorescence staining of CVB3 mouse heart coronary artery sections after 4 weeks of saline or aFAP-CAR T cell treatment. (D, E) Statistics of body weight and ejection fraction of each group. (F, G) Quantification of ventricular fibrosis and FAP expression. (H, I, and J) Staining of CD68 + , LY6C + , MPO + cells in heart sections after different treatments. (K, L, and M) Staining of CD3 + , CD4 + , and CD8 + T cells in heart sections after different treatments. Representative images of two independent experiments, with similar results. Scale bar, 100 pm. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; NS, not significant; one-way ANOVA between groups.

[0122] We also performed the same test in a viral myocarditis model (A in Figure 7 ). Similarly, we injected anti-aFAP-CAR T cells immediately after the CVB3 model was established (day 7) and again after 2 weeks (day 21). Four weeks after the mouse model was established, the mice in each group were measured and sampled. The results were similar to the trend in the EAM group Figure 7 . The above results all indicate that aFAP-CAR T has excellent effects on fibrosis in the chronic phase of myocarditis.

[0123] After 42 days of treatment, the mice were sacrificed, and their blood was collected for hematological and blood biochemical analysis. This study compared the blood biochemical indicators of liver function (alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and globulin (GLB)) and kidney function (creatinine (Cr)) between the saline group and the aFAP-CAR T NPs group. The levels of uric acid (UA), total protein (TP), and urea in the aFAP-CAR T NPs group were within the normal range, and the difference was not statistically significant. The results showed that aFAP-CAR T therapy did not cause any significant systemic side effects Figure 7Figure 6. Anti-aFAP-CAR T cell therapy had no histological impact on non-cardiac organs or tissues such as heart, liver, spleen, lung, kidney, intestine and skin (A). Figure 7 Figure 6. Anti-aFAP-CAR T cell therapy had no histological impact on non-cardiac organs or tissues such as heart, liver, spleen, lung, kidney, intestine and skin (A).

[0124] Figure 7 Figure 7. In vivo biological safety evaluation results, (A) Blood biochemical indicators of mice after treatment. (B) H&E staining of different tissue sections of mice treated with normal saline and aFAP-CAR T cells for 4 weeks. Representative images of two independent experiments, similar results. Scale bar, 100 pm.

[0125] In summary, the present application found that the expression of FAP was positively correlated with the degree of fibrosis, and the FAP-specific CAR T cells constructed had excellent treatment effect on fibrosis in the chronic phase of myocarditis.

[0126] The applicant states that the above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by any person skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. The application of a CAR-T cell targeting FAP in the preparation of a drug for treating chronic myocarditis and cardiac fibrosis, characterized in that, The CAR-T cells targeting FAP contain CAR molecules; The amino acid sequence of the CAR molecule is shown in SEQ ID NO.

10.

2. The application according to claim 1, characterized in that, The amino acid sequence of the extracellular recognition domain of the CAR molecule is selected from: The amino acid sequence is shown in SEQ ID NO.

2.

3. The application according to claim 2, characterized in that, The CAR molecule comprises a signal peptide, an extracellular recognition domain, a transmembrane domain, and a signal transduction domain connected in series.

4. The application according to claim 3, characterized in that, The transmembrane structural domain is the CD28 hinge region and the CD28 transmembrane region.

5. The application according to claim 3, characterized in that, The signal conduction structure domain is the CD28 signal inner domain and the CD3z inner domain.

6. The application according to claim 3, characterized in that, The signal peptide is also labeled with a FLAG tag between itself and the extracellular recognition domain.

7. The application according to claim 1, characterized in that, The CAR-T cells were prepared using a method comprising the following steps: An expression vector was constructed, wherein the expression vector contains a nucleic acid molecule encoding a CAR molecule targeting FAP; The expression vector and packaging helper plasmid were co-transfected into mammalian cells to obtain recombinant lentivirus; The recombinant lentivirus was transfected into T cells for expression to obtain CAR-T cells, which expressed CAR molecules targeting FAP.

8. The application according to claim 7, characterized in that, The expression vector is a viral vector.

9. The application according to claim 8, characterized in that, The viral vector is any one of a lentiviral vector, a retroviral vector, or an adeno-associated virus vector.

10. The application according to claim 9, characterized in that, The viral vector is a retroviral vector.

11. The application according to claim 7 or 8, characterized in that, The nucleic acid molecule encoding the CAR molecule targeting FAP is shown in SEQ ID NO.9.

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