Application of FAP-targeted CAR-T cell in preparation of medicine for treating cardiac fibrosis in chronic period of myocarditis

Abstract

The invention provides an application of a CAR-T cell targeting FAP in preparation of a medicine for treating cardiac fibrosis in a myocarditis chronic stage. According to the present invention, it is found that the FAP specific CAR-T cell can effectively target the cells expressing the mouse FAP protein, the CAR-T cell can effectively remove the cells expressing the mouse FAP protein after being activated, and the chimeric antigen receptor T cell can effectively alleviate the cardiac fibrosis of the mouse at the chronic period of myocarditis, and can recover the function after the inflammation injury. The invention develops a CAR-T therapy for myocardial fibrosis caused by the chronic period of myocarditis, the chimeric antigen receptor T cell expressing the targeted fibroblast activating protein can recognize activated myocardial fibroblasts, and the cardiac fibrosis after inflammation injury can be obviously relieved and the function can be recovered.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an application of FAP-targeted CAR-T cells in the preparation of a drug for treating cardiac fibrosis in the chronic phase of myocarditis. Background Art

[0002] Chronic inflammation in patients with myocarditis can lead to myocardial fibrosis. After injury, activated fibroblasts secrete a large amount of fibrotic factors and extracellular matrix, which accumulate in the myocardial interstitium and perivascular spaces to reshape the myocardium. Myocardial fibrosis is a remodeling process of the myocardial interstitium, characterized by abnormal proliferation of myocardial interstitial fibroblasts, excessive accumulation of collagen fibers, a significant increase in the concentration and volume fraction of collagen per unit mass of myocardium, and an imbalance in the proportion and abnormal distribution of various types of collagen. The reason is that myofibroblasts excessively deposit extracellular matrix proteins under chronic inflammatory response, leading to scar formation and tissue sclerosis, gradually impaired cardiac function, and ultimately heart failure. However, most of the drugs used to intervene in myocarditis clinically focus on anti-heart failure or arrhythmia, rather than the fibrosis process, and cannot prevent 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 the renin-angiotensin-aldosterone system pathway and the TGF-β pathway. Myocardial damage caused by inflammation is one of the important causes of myocardial fibrosis. During the chronic inflammatory process, a large number of immune cells, mainly monocytes and macrophages, gather and infiltrate, causing myocardial damage, and at the same time secrete a large number of proinflammatory mediators, including tumor necrosis factor-α, interleukin-6, and interleukin-1, forming a chronic inflammatory microenvironment. In this cellular environment, cytokines and growth factors are released in large quantities, including members of the transforming growth factor-β family and Wingless / Int-1, which are the main effectors of the fibrotic process. TGF-β and Wnt1 bind to stem cell surface receptors and initiate downstream signaling, ultimately leading to Smad2 / 3 and CBP / β-Catenin transcriptional regulators, respectively. This leads to upregulation of target gene expression, further enhancing the differentiation of myofibroblasts and the production and secretion of collagen, laminin, and fibronectin, thereby 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, and thus treating fibrosis. However, activated myofibroblasts do not stop there, and the fibrosis process will not be interrupted. Therefore, it is urgent to develop new therapeutic strategies to target fibrosis in the chronic stage of myocarditis.

[0005] Chimeric antigen receptor T cell therapy (CAR-T) has made breakthrough progress in the treatment of tumors, autoimmunity and leukemia. It extracts T cells from the patient's immune system, cultures and modifies T cells in vitro to enable them to recognize and attack specific cancer cells, and then injects the modified T cells back into the patient's body. The immune response of T cells to cancer cells will eliminate cancer cells. The U.S. Food and Drug Administration has approved CAR-T cell therapy for patients with certain forms of leukemia and lymphoma. 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, using CAR-T cells to target non-cancerous cells is a new attempt. Jonathan Epstein's team has achieved quantitatively good results in the treatment of heart failure caused by fibrosis using CAR-T cell therapy. Studies have shown that CAR-T cell therapy can effectively inhibit the myocardial fibrosis process in AngII-induced heart failure model mice. However, it is currently unclear whether CAR-T therapy can be applied to myocardial fibrosis induced by chronic inflammation.

[0006] Therefore, there is an urgent need to develop a CAR-T therapy targeting myocardial fibrosis caused by the chronic stage of myocarditis. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a CAR-T cell targeting FAP for use in the preparation of a drug for treating cardiac fibrosis in the chronic phase of myocarditis. The present invention found that FAP is a potential candidate for targeting cardiac fibroblasts in the pathology of myocarditis, and developed a Fap-specific CAR-T cell (αFAP-CAR-T), which has excellent therapeutic effects on fibrosis in the chronic phase of myocarditis.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides the use of a substance using the Fap gene and / or FAP protein as a target in the preparation of a drug for treating cardiac fibrosis in the chronic phase of myocarditis.

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

[0011] Preferably, the substance targeting FAP protein 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 the Fap gene, or a vector of a Fap gene knockdown or knockout system.

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

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

[0015] The present invention found that FAP-specific CAR-T cells can effectively target cells expressing mouse FAP protein, and after being activated, CAR-T cells can also effectively eliminate cells expressing mouse FAP protein.

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

[0017] The present invention develops a CAR-T therapy for myocardial fibrosis caused by chronic myocarditis. The specific genes of chronic myocarditis fibrosis and endogenous targets of cardiac fibrosis are determined by RNA sequencing data of myocarditis patients, dilated cardiomyopathy patients and healthy people. Chimeric antigen receptors for targets are expressed by constructing CAR-T. Subsequently, experimental autoimmune myocarditis (EAM) model and viral myocarditis model (CVB9) were constructed to simulate chronic myocardial fibrosis associated with inflammation. CAR-T was regularly injected into myocarditis mice intravenously to study its effect on treating myocardial fibrosis in the chronic stage of myocarditis. According to the results of the study 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 with chronic myocarditis, and its expression is related to the degree of cardiac fibrosis. Therefore, FAP was selected as a marker for chronic myocarditis fibrosis. Chimeric antigen receptor T cells expressing targeted fibroblast activation protein (αFAP-CAR-T) can recognize activated cardiac fibroblasts and significantly reduce cardiac fibrosis and restore function after inflammatory injury in mice. This approach effectively reduced cardiac fibrosis in the chronic phase of myocarditis in mice and restored function after inflammatory injury. These findings provide evidence for the development of CAR-T in the treatment of cardiac immune-related diseases.

[0018] Preferably, the CAR molecule targeting FAP protein specifically binds to 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 in 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: a signal peptide, an extracellular recognition domain, a transmembrane domain and a signal transduction domain connected in series.

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

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

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

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

[0025] Preferably, the CAR-T cells are 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 the packaging helper plasmid into mammalian cells to obtain a recombinant lentivirus;

[0028] The recombinant lentivirus is transfected into T cells for expression to obtain CAR-T cells, which express CAR molecules 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 the CAR molecule targeting FAP includes that shown in SEQ ID NO.9.

[0032] Preferably, the amino acid sequence of the FAP-targeting CAR molecule includes that shown in SEQ ID NO.10.

[0033] In a second aspect, the present invention provides a chimeric antigen receptor targeting FAP, wherein the chimeric antigen receptor specifically binds to the FAP protein expressed by cardiac fibroblasts, and the amino acid sequence of the 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 in the amino acid sequence as shown in SEQ ID NO.2, which has the function of binding to FAP.

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

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

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

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

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

[0040] In a third aspect, the present invention provides a nucleic acid molecule encoding the chimeric antigen receptor targeting FAP as described in the second aspect.

[0041] In a fourth aspect, the present invention provides an expression vector comprising at least one copy of the nucleic acid molecule described in 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 invention provides a recombinant lentivirus, which is prepared by a preparation method comprising the following steps: co-transfecting the viral vector described in the fourth aspect with a packaging helper plasmid into mammalian cells to obtain the recombinant lentivirus.

[0045] In a sixth aspect, the present invention provides a chimeric antigen receptor T cell targeting FAP, wherein the chimeric antigen receptor T cell is prepared by a preparation method comprising the following steps: transfecting the recombinant lentivirus described in the fifth aspect into a T cell for expression to obtain the chimeric antigen receptor T cell, wherein the cell expresses the chimeric antigen receptor targeting FAP described in the second aspect.

[0046] In the present invention, the method for preparing the chimeric antigen receptor T cells targeting FAP comprises:

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

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

[0049] (3) Introducing the recombinant lentivirus in step (2) into T cells to prepare T cells expressing chimeric antigen receptor targeting FAP.

[0050] In a seventh aspect, the present invention provides a pharmaceutical composition, comprising the chimeric antigen receptor T cell targeting FAP as described in the sixth aspect.

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

[0052] In an eighth aspect, the present invention provides the use of the chimeric antigen receptor T cell targeting FAP described in the sixth aspect and / or the pharmaceutical composition described in 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 invention has the following beneficial effects:

[0054] (1) The present invention found that the expression of FAP is positively correlated with the degree of fibrosis and that 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 by the present invention can effectively target and lyse cells expressing mouse FAP protein, and αFAP-CAR T has excellent therapeutic effect on fibrosis in the chronic stage of myocarditis. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0057] Figure 2 This is an immunohistochemical staining image of a human myocardium sample.

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

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

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

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

[0062] Figure 7 This is a diagram showing the results of the in vivo biosafety evaluation. DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0064] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0065] Example 1

[0066] 1. Analysis of human heart expression data

[0067] Human heart samples were obtained from patients undergoing heart transplantation ( Table 1 ). All collected human hearts were cardioprotected using in situ cardioplegia. In vivo echocardiography was performed before tissue collection to allow real-time assessment of in vivo structure and function. The clinical study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Review Board of the Fuwai Hospital. All participants provided written informed consent before participating in the study. Tissue specimens were frozen with liquid nitrogen and stored at −80°C before use.

[0068] Table 1

[0069]

[0070]

[0071] Variables are presented as frequencies (percentages) and mean ± SD. Bold italics indicate statistical significance. HTx, heart transplant. MACE: major adverse cardiovascular event. 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-seq analysis, library preparation was performed using the Illumina truSeq stranded mRNA kit, followed by a Nugen Ovation amplification kit. The resulting FASTQ files were assessed for quality control using the FastQC program. The FASTQ files were aligned to the human reference genome (hGRC37 / Hg19) using STAR aligner46. Duplicate reads were marked using the MarkDuplicates program of Picard tools. Read counts per gene for Ensembl (v.75) gene annotations were calculated using the R package and duplicate reads were removed. Gene counts expressed in counts per million (CPM) were first normalized using the TMM method in the edgeR R package, and genes with CPM < 1 in 25% of the samples were removed and considered as lowly expressed genes. Data were transformed using the VOOM function of the limma R package. Differential gene expression was analyzed using linear models of the limma package.

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

[0074] The Animal Ethics and Welfare Committee of Shenzhen Hospital, Fuwai Hospital, Chinese Academy of Medical Sciences approved all animal experiments involved in the present invention. Six-week-old male Balb / c mice were purchased from Guangzhou Jicui Pharmaceutical Co., Ltd. The mice were adapted for at least one week before the experiment and 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), and the α-MyHC peptide was emulsified with complete Freund's adjuvant (Sigma, F5881, 1:1, weight ratio) on days 0 and 7. On the 14th day after injection of α-MyHC, EAM mice were successfully established. Method for modeling viral myocarditis in mice: 6-week-old male inbred Balb / c mice weighing (15-19) g were selected and divided into experimental and normal groups. After routine disinfection of the injection site, 10 5 That is, 0.1 mL of CVB3 with 100 TCID 50 / 0.1 mL was injected intraperitoneally, and 0.1 mL of 0.1 mmol / L phosphate buffer was injected intraperitoneally in the normal group. The CVB3 mouse model was successfully established on the 7th day of virus inoculation.

[0075] 3. Plasmid construction

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

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

[0082] ATTGAGTTCATGTACCCTCCGCCTTACCTAGACAACGAGAGGAGCAATGGAACTATTATTCACATAAAAGAGAAACATCTTTGTCATACTCAGTCATCTCCTAAGCTGTTTTGGGCACTGGTCGTGGTTGCTGGAGTCCTGTTTTGTTATGGCTTG CTAGTGACAGTGGCTCTTTGTGTTATCTGGACAAATAGTAGAAGGAACAGACTCCTTCAAAGTGACTACATGAACATGACTCCCCGGAGGCCTGGGCTCACTCGAAAGCCTTACCAGCCCTACGCCCCTGCCAGAGACTTTGCAGCGTACCGCCCC.

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

[0084] IEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFCYGLLVTVALC VIWTNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRP

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

[0086] AGAGCAAAATTCAGCAGGAGTGCAGAGACTGCTGCCAACCTGCAGGACCCCAACCAGCTCTACAATGAGCTCAATCTAGGGCGAAGAGAGGAATATGACGTCTTGGAGAAGAAGCGGGCTCGGGATCCAGAGATGGGAGGCAAACAGCAGAGGAGGAGGAACCCCCAGGA AGGCGTATACAATGCACTGCAGAAAGACAAGATGGCAGAAGCCTACAGTGAGATCGGCACAAAAGGCGAGAGGCGGAGAGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGCACTGCCACCAAGGACACCTATGATGCCCTGCATATGCAGACCCTGGCCCCTCGC.

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

[0088] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQ EGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR.

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

[0090] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACTACAAAGACGATGACGACAAG.

[0091] The amino acid sequence of the signal peptide and 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 chimeric antigen receptor targeting mouse FAP: SEQ ID NO.10:

[0096] MALPVTALLLPLALLLHAARPDYKDDDDKQVQLKESGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTADKRLELVATTNNNGGVTYYPDSVKGRFTISRDNAKNTLYLQMSSLQSEDTAMY YCARYGYYAMDYWGQGISVTVSSGGGGSGGGGSSGGGSDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTVKISRVEA EDLGVYYCFQGSHVPYTFGGGTKLEIKTRIEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYA PARDFAAYRPRAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR.

[0097] 4. Retrovirus Preparation

[0098] The retrovirus encoding the CAR molecule was made as previously reported [PMID: 32015548]. Briefly, the transfer vector and the eco-pCL-Eco packaging vector were mixed at a ratio of 3:1 and then used HEK293T cells were transfected with transfection reagent (Mirus) according to the manufacturer's instructions. Supernatants containing retrovirus were collected 48 and 72 hours after transfection, filtered, snap-frozen and stored at -80°C before use.

[0099] 5. Preparation of mouse CAR T cells

[0100] On day 0, mouse CAR T cells were generated as previously reported [PMID: 32015548]. Briefly, spleen cells were collected from 6-8 week 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 antibody (eBioscience). Activated mouse T lymphocytes were transduced with retroviral supernatant using plates coated with retronectin (recombinant human fibronectin) [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 lines expressing mouse FAP

[0102] Retrovirus encoding mouse Fap was prepared using a two-dimensional packaging vector (RDF114) and transduced into a human lymphoma cell line (Daudi) using spin-ligation. The expression of mFap was detected by staining the surface of mouse FAP with DNGFR and anti-mouse FAP monoclonal antibody (clone 73.3, Millipore).

[0103] 7. Flow cytometric analysis

[0104] For cell surface staining, cells were incubated with antibodies for 15 min at room temperature or 30 min at 4°C. The following antibodies used 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 antibodies were from BD Bioscience: PE-conjugated anti-mouse CD3 (clone 500A2). Flow cytometric data were acquired on a NovocyteQuanteon (Agilent) using NovoExpress software and analyzed using FlowJo software (v10.6.2, TreeStar).

[0105] 8. Histological examination

[0106] Heart tissues from mouse and human samples were collected, fixed overnight in 4% (v / v) formalin solution, embedded in paraffin, and cut into 5 μm thick sections. The sections were then subjected to H&E, immunohistochemistry, and immunohistochemical fluorescence staining for histopathological evaluation. H&E and immunohistochemical images were examined by digital microscopy. 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 χ2 test. Spearman correlation coefficients were used to test the associations 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 analyses were used to test the association between the time to outcome events 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 identifies endogenous proteins expressed by activated cardiac fibroblasts caused by myocarditis, which can be designed (or genetically modified) 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 the results were analyzed for differential gene expression. The clinical manifestations of the two groups of patients were similar, including gender, symptoms / heart transplant age, and arrhythmias. We found that the serum NT-proBNP level in the DCM group was higher than that in the myocarditis group (3823.3±1734.9vs1622.2±264.0fmol / ml, P<0.001). In addition, the myocarditis group had a higher ventricular septum thickness, but lower LVEDD and LVEF.

[0111] Figure 1Figure 1 shows the single-cell sequencing analysis of human myocardial samples, including (A) heat map of changes in gene expression (fold change) in cardiac fibroblasts from DCM and MYO patients compared with non-failing hearts, n = 15 DCM hearts, 4 MYO hearts. (B) Umap of cardiac fibroblast clusters under heart failure and normal conditions, data from previous studies (GSE145154). (C) Violin map of FAP expression in fibroblast clusters.

[0112] Figure 2 Figure 1. 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 hearts and MYO human left ventricular free wall sections. Representative images of two independent experiments showing similar results. (E-F) Quantification of ventricular fibrosis and FAP expression. (G) Correlation of fibrosis percentage with FAP expression.

[0113] The results showed that several fibroblast-specific genes were upregulated in the myocardium of patients with DCM or myocarditis compared with controls and non-failing donor hearts ( Figure 1 (A). Among these genes, FAP had the most significant expression and up-regulation fold change. FAP is a cell surface glycoprotein that has previously been shown to be expressed during embryonic development at sites of active tissue remodeling (such as during wound repair), during tissue fibrosis, and in various tumors, but is only expressed at low levels in most normal adult tissues in mice and humans. FAP expression has previously been observed in the human heart after acute myocardial infarction injury26,28. In line with previous studies on the profiling of fibroblasts in the human heart (including normal conditions) (GEO:GSE145154)29, we found that FAP is only expressed in a specific fibroblast subset called myofibroblasts (FB_7) ( Figure 1 We further found that in the left ventricular tissue of human failing myocarditis and DCM, cardiac fibroblasts (but not cardiomyocytes) strongly expressed FAP, while FAP expression was very low in normal human hearts ( Figure 2 In addition, the expression of FAP was positively correlated with the degree of fibrosis ( Figure 2 Middle G). Therefore, FAP is a potential candidate for targeting cardiac fibroblasts in the pathology of myocarditis.

[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 clone 73.3 scFv sequence, mouse CD28 and CD3 endodomains, and a FLAG tag as a detection marker. We expressed this CAR molecule in primary mouse T cells using retroviral transduction technology.

[0115] Figure 3 Schematic diagram of the construction of mouse Fap-specific CAR T cells; (A) is the construction and verification of mouse FAP-specific CAR T cells, (n=3). (B) Overexpression of mouse Fap gene and FACS sorting of mFap-positive target cells. (C) Schematic diagram of functional experiment to verify the specificity of mouse Fap-specific CAR T cells.

[0116] Figure 4 The validation diagram of mouse Fap-specific CAR T cells; (D) CD4 + and CD8 + Expression of CD69 activation marker on T cells, n=3 (Ctrl), n=4 (αFAP-CAR Ts). (E) Percent lysis of mFap positive or negative targets when co-cultured with Ctrl or αFAP-CAR T cells, n=3 (Ctrl), n=4 (αFAP-CAR Ts). (F) Cytokine production by Ctrl or αFAP-CAR T cells when co-cultured with mFap positive or negative targets.

[0117] The results showed that the CAR molecule was efficiently expressed ( Figure 3 To verify the function of the expressed CAR molecule, we engineered a human cell line that ectopically expressed the mouse Fap (mFap) gene and sorted the surface Fap-positive cells by 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 C), and untransduced T cells were used as control (Ctrl). We found that Fap-specific CAR T cells could be clearly activated by mFap-positive targets based on CD69 expression. In contrast, Ctrl T cells remained unactivated ( Figure 4 In addition, activation of Fap-specific CAR T cells can effectively eliminate mFAP-positive target cells. However, there is no mFAP negative control ( Figure 4Middle E). Fap-specific CAR T cells can also produce a large amount of cytokines (TNFa, IFNg and IL-2) after co-culture with mFap-positive target cells ( Figure 4 In summary, these experiments demonstrate that our Fap-specific CAR T cells can effectively target and lyse cells expressing mouse Fap protein.

[0118] Figure 5 The experimental results of αFAP-CAR T cell targeted therapy for cardiac fibrosis in EAM model mice are shown in Figure 1, where (A) Schematic diagram of the experiment of αFAP-CAR T cells targeting cardiac fibroblasts. (B) M-mode echocardiograms and digital photos of mice treated with saline (top), EAM (middle) or αFAP-CAR T cells (bottom). (C) After 4 weeks of treatment with saline or αFAP-CAR T cells, coronary artery sections of EAM mice were subjected to H&E staining, fibrosis Masson trichrome staining (blue) and immunofluorescence staining of FAP (red), CD68 (red), LY6C (green), MPO (yellow), CD3 (red), CD4 (green) and CD8 (yellow). (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) CD68 in heart sections after different treatments + LY6C + 、MPO + (K, L and M) CD3 in heart sections after different treatments + 、CD4 + and CD8 + Staining of T cells. Representative images of two independent experiments with similar results. Scale bars, 100 μm. ****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 established an autoimmune myocarditis (EAM) mouse model and a viral myocarditis model. Six weeks after Myhc sensitization of Balb / c mice, cardiac fibrosis was already severe. In this EAM model, the presence of FAP in activated cardiac fibroblasts was confirmed.

[0120] Immunohistochemistry showed that FAP was not detected in the hearts of the control group, but was significantly expressed in the activated fibroblast tissue samples of EAM mice six weeks later ( Figure 6To eliminate FAP-expressing cardiac fibroblasts in the chronic phase of EAM, we injected anti-αFAP-CAR T cells immediately after the establishment of the EAM model (day 14) and again 2 weeks later (day 28). Figure 6 We closely monitored the body weight and cardiac function measurements of the mice. Four weeks after the mouse model was established, the mice in each group were measured and sampled. The results showed that the body weight of mice treated with anti-αFAP-CAR T cells remained the same, similar to that of the control group. In addition, their cardiac function was comparable to that of the control group and better than that of the EAM group ( Figure 6 In addition, the degree of cardiac fibrosis in EAM mice was significantly reduced ( Figure 6 C, F). All mice treated with anti-αFAP-CAR T cells showed significantly reduced fibrosis compared with the control group. Our results show that anti-αFAP-CAR T cells can effectively reduce cardiac fibrosis in EAM mice. In addition, the treated mice fully restored cardiac systolic and diastolic function ( Figure 6 We also performed immunofluorescence staining on normal tissues and myocarditis tissues of mice to further confirm our findings. The results showed that the red fluorescence of CD68, the green fluorescence of LY6C, and the yellow fluorescence of MPO were significantly more obvious in the myocarditis samples compared with the αFAP-CAR T group. In addition, these three fluorescences were almost undetectable in the control group ( Figure 6 Compared with the other two groups, the red, green, and yellow fluorescence levels of the αFAP-CAR T group were significantly higher, representing CD3 + 、CD4 + and CD8 + T cells ( Figure 6 (C, KM).

[0121] Figure 6The experimental results of αFAP-CAR T cell targeted therapy for cardiac fibrosis in CVB3 model mice are shown in Figure 1. (A) Schematic diagram of the experiment of αFAP-CAR T cells targeting cardiac fibroblasts. (B) M-mode echocardiograms and digital photos of mice treated with saline (top), CVB3 (middle) or αFAP-CAR T cells (bottom). (C) After 4 weeks of treatment with saline or αFAP-CAR T cells, coronary artery sections of CVB3 mice were stained with H&E, fibrosis Masson's trichrome staining (blue) and immunofluorescence staining of FAP (red), CD68 (red), LY6C (green), MPO (yellow), CD3 (red), CD4 (green) and CD8 (yellow). (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) CD68 in heart sections after different treatments + LY6C + 、MPO + (K, L and M) CD3 in heart sections after different treatments + 、CD4 + and CD8 + Staining of T cells. Representative images of two independent experiments with similar results. Scale bars, 100 μm. ****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 ( Figure 7 Similarly, we injected anti-αFAP-CAR T cells immediately after the CVB3 model was established (day 7) and injected them again 2 weeks later (day 21). Four weeks after the mouse model was established, the mice in each group were measured and samples were collected. The results showed the same trend as the EAM group ( Figure 7 ). The above results all indicate that αFAP-CAR T has excellent therapeutic effect on fibrosis in the chronic stage of myocarditis.

[0123] After 42 days of treatment, the mice were killed and their blood was collected for hematological and blood biochemical analysis. The study compared the blood biochemical indicators of liver function indicators (alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and globulin (GLB)) and renal function indicators (creatinine (Cr)) between the normal saline group and the αFAP-CAR T NPs group. The levels of uric acid (UA), total protein (TP) and urea in the αFAP-CAR T NPs group were within the normal range, and there was no statistically significant difference. The results showed that αFAP-CAR T therapy did not cause any obvious systemic side effects ( Figure 7Middle A). Anti-αFAP-CAR T cell therapy had no histological effects on non-cardiac organs or tissues such as the heart, liver, spleen, lung, kidney, intestine, and skin ( Figure 7 This is consistent with previous studies using anti-αFAP-CAR T cells to target tumor stromal cells or myocardial fibrosis in HCM and DCM without observing any toxicity.

[0124] Figure 7 Figure 1 shows the results of in vivo biosafety evaluation, where (A) blood biochemical indicators of mice after treatment. (B) H&E staining of different tissue sections of mice treated with saline and αFAP-CAR T cells for 4 weeks. Representative images of two independent experiments with similar results. Scale bar, 100 μm.

[0125] In summary, the present invention found that the expression of FAP was positively correlated with the degree of fibrosis, constructed FAP-specific CAR T cells, and verified that they had excellent therapeutic effects on fibrosis in the chronic stage of myocarditis.

[0126] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. Use of substances with Fap gene and / or FAP protein as targets in the preparation of drugs for treating cardiac fibrosis in the chronic phase of myocarditis.

2. The use according to claim 1, characterized in that: The substance that targets the Fap gene is an inhibitor that reduces the activity and / or expression of the Fap gene; Preferably, the substance targeting FAP protein 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; Preferably, the inhibitor comprises: any one or a combination of two of a small molecule compound, an interfering RNA vector expressing the Fap gene, or a vector of a Fap gene knockdown or knockout system.

3. The use according to claim 2, characterized in that: The vector comprises: any one of adenovirus, adeno-associated virus, lentivirus, plasmid, liposome or nanoparticle, or a combination of at least two thereof.

4. The use according to claim 2, characterized in that: The CAR cells targeting FAP protein include any one of CAR-T, CAR-NK, CAR-NKT, CAR-M, CAR Treg or CAR-γδT.

5. The use according to claim 2, characterized in that: The CAR molecule targeting 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: 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 in the amino acid sequence as shown in SEQ ID NO.2, which has the function of binding to FAP.

6. The use according to claim 2 or 5, characterized in that: The CAR molecule comprises: a signal peptide, an extracellular recognition domain, a transmembrane domain and a signal transduction domain are sequentially connected in series; Preferably, the transmembrane domain is the CD28 hinge region and the CD28 transmembrane region; Preferably, the signal transduction domain is the CD28 signaling domain and the CD3z domain; Preferably, there is a FLAG tag between the signal peptide and the extracellular recognition domain; Preferably, the CAR molecule comprises the amino acid sequence shown in SEQ ID NO.

10.

7. The use according to claim 4, characterized in that: The CAR-T cells are prepared by a preparation method comprising the following steps: Constructing an expression vector containing a nucleic acid molecule encoding a CAR molecule targeting FAP; Co-transfecting the expression vector and the packaging helper plasmid into mammalian cells to obtain a recombinant lentivirus; The recombinant lentivirus is transfected into T cells for expression to obtain CAR-T cells, which express CAR molecules targeting FAP.

8. The use according to claim 7, characterized in that: The expression vector includes a viral vector; Preferably, the viral vector comprises any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector, preferably a retroviral vector.

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

9.

10. The use according to any one of claims 7 to 9, characterized in that: The amino acid sequence of the FAP-targeting CAR molecule includes that shown in SEQ ID NO.10.

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