Intervention target for advanced fibrosis of ischemic heart disease and use thereof

Abstract

The present invention relates to the field of biomedicines. Disclosed are an intervention target for advanced fibrosis of an ischemic heart disease and a use thereof. In the present invention, it was found that a CD248 protein is only specifically and highly expressed in cardiac fibroblasts in an ischemic injury area, and is expressed at lower levels in other normal tissue cells, and therefore, the CD248 protein can be used as an intervention target for advanced fibrosis of an ischemic heart disease to effectively inhibit excessive cardiac fibrosis, and the safety is high. According to the present invention, an RNA fragment, a monoclonal antibody and a CAR-T cell targeting CD248 are prepared; the RNA fragment can inhibit CD248 gene expression; the monoclonal antibody has good affinity to the CD248 protein, and can effectively inhibit the activation of fibroblasts; and the CAR-T cell has better infiltration into a heart ischemia injury area, and can kill CD248+ fibroblasts.

Description

A target for intervention of late-stage fibrosis in ischemic heart disease and its application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an intervention target for late-stage fibrosis of ischemic heart disease and an application thereof. Background Art

[0002] In patients with ischemic heart disease, the degree of cardiac fibrosis is a key predictor of poor prognosis, with its severity being associated with increased rates of hospitalization, mortality, and adverse cardiac events. Acute myocardial infarction (AMI) is the most common and fatal acute myocardial ischemic event in clinical practice. While emergency interventional procedures may allow the diseased coronary arteries of some patients to be recanalized within a short period of time, these patients are still susceptible to ischemia / reperfusion injury. Fibroblasts are the primary stromal cell type in the heart and play a crucial role in maintaining cardiac structure and regulating its function. A growing body of research indicates that cardiac fibroblasts are a highly heterogeneous cell type, with different subpopulations of fibroblasts functioning differently under physiological and pathological conditions. In the early stages of myocardial infarction (2-7 days), fibroblasts, in response to damaging stimuli such as hypoxia and inflammation, are activated to transdifferentiate into myofibroblasts, which migrate to the site of injury and synthesize and secrete collagen matrix, promoting the formation of fibrous scars, thereby repairing damaged myocardial tissue and preventing cardiac rupture. However, in the late stages of myocardial infarction (after 10 days), the continued activation of profibrotic signals leads to excessive production of collagen matrix, increasing cardiac stiffness, decreasing cardiac compliance, and affecting cardiac electrical activity, ultimately leading to the detrimental consequences of heart failure. Therefore, inhibiting excessive fibroblast activation in the late stages of myocardial infarction has positive implications.

[0003] Through the preliminary research of the present invention team, we found that different subpopulations of fibroblasts have different effects at different stages of myocardial infarction. However, due to the lack of reliable biomarkers, we still lack an understanding of the specific role of fibroblasts in cardiac homeostasis and at different phases after myocardial infarction. Therefore, it is still impossible to screen out the fibroblast subpopulations that play a key role in cardiac remodeling in the late stage of myocardial infarction. On the other hand, the anti-fibrosis targets and strategies currently developed and applied in basic and clinical research are relatively broad and cannot target specific pathological fibroblast subpopulations, resulting in poor anti-fibrosis treatment effects and possible cardiac toxicity. The rapid development of single-cell sequencing technology in recent years has provided us with a powerful tool to understand the heterogeneity of cardiac fibroblasts and find reliable biomarkers and therapeutic targets. With the help of single-cell sequencing, we can analyze the dynamic changes of cardiac fibroblast subpopulations at different phases / regions after myocardial infarction, and then find the fibroblast subpopulations that play a key role in cardiac pathological remodeling in the late stage of myocardial infarction, so as to improve the late stage of adverse interstitial remodeling without affecting the early scar repair, which is crucial to solving the problem of cardiac fibrosis.

[0004] After myocardial infarction, fibroblasts undergo various changes, including transdifferentiation, dedifferentiation, apoptosis, and senescence. These different fates of fibroblasts are closely involved in regulating cardiac remodeling after myocardial infarction. Fibroblasts, particularly myofibroblasts, can overproduce extracellular matrix in the long term after myocardial infarction, promoting cardiac interstitial fibrosis. Inhibiting myofibroblast function or eliminating specific myofibroblast subsets are potential interventions for fibrosis.

[0005] The recent development of chimeric antigen receptor (CAR)-T cell technology in the oncology field enables T cells to target and kill pathological cells carrying specific antigens. CARs combine intracellular T cell signaling and co-stimulatory molecules with single-chain variable regions of specific antigens. The advent of CAR-T cell technology has revolutionized the treatment of refractory acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, for the application of CAR-T technology in the treatment of cardiac fibrosis, identifying intervention targets that are specifically expressed in pathological fibroblasts and have low or no expression in other cells and tissues is crucial to ensuring its safety and effectiveness.

[0006] Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a target for intervention of late-stage fibrosis in ischemic heart disease and its application. First, the present invention uses single-cell sequencing technology to discover a group of CD248 that plays a key role in late-stage cardiac fibrosis in ischemic heart disease. + Fibroblasts; CD248 protein is specifically and highly expressed only in cardiac fibroblasts in the ischemic injury area, and is expressed at a lower level in other normal tissue cells. Therefore, using CD248 as a target for intervention in late fibrosis of ischemic heart disease can not only effectively inhibit excessive cardiac fibrosis, but also has high safety. Secondly, the present invention prepares RNA fragments, monoclonal antibodies and CAR-T cells targeting CD248; among them, small interfering RNA fragments can inhibit fibroblast activation and collagen matrix synthesis under TGF-β stimulation; monoclonal antibody IgG78 has good affinity for CD248 protein and can effectively inhibit fibroblast activation; CAR-T cells have good infiltration into the ischemic injury area of ​​the heart and can effectively reduce CD248 in the ischemic area. + Therefore, the monoclonal antibodies and CAR-T cells of the present invention can significantly reduce cardiac interstitial fibrosis and improve cardiac contractile function.

[0008] The specific technical solutions of the present invention are:

[0009] In the first aspect, the present invention provides a target for intervention of late fibrosis of ischemic heart disease, wherein the intervention target is CD248 gene, CD248 protein or CD248 +Fibroblasts.

[0010] As described in the background technology of this application, in the late stage of ischemic heart disease (after 10 days), the continuous activation of pro-fibrotic signals will lead to excessive cardiac fibrosis, and ultimately bring about the malignant consequence of heart failure. Therefore, it is necessary to inhibit the above-mentioned fibrosis process in the late stage of ischemic heart disease. Through the preliminary research of the present invention team, we found that the degree of fibrosis of different subpopulations of fibroblasts in different stages of ischemic heart disease is different. To this end, the present invention innovatively uses single-cell sequencing technology to analyze the dynamic changes of cardiac fibroblast subpopulations in different phases / regions, and finally discovered a group of CD248 that plays a key role in cardiac fibrosis in the late stage of ischemic heart disease. + Fibroblasts; CD248 protein is specifically and highly expressed only in activated cardiac fibroblasts, especially in the late stage of ischemic heart disease, and is one of the main factors leading to excessive cardiac fibrosis; in addition, the expression level of CD248 protein in normal tissue cells is low, so it has high safety as an intervention target.

[0011] In a second aspect, the present invention provides a CD248 gene, a CD248 protein or a CD248 + Application of fibroblasts as intervention targets in the preparation of anti-fibrotic drugs for late ischemic heart disease.

[0012] Using CD248 as an intervention target, we can specifically prepare drugs that can inhibit CD248 gene expression, inhibit CD248 protein activity, reduce the number of CD248 proteins, or kill CD248. + fibroblast-inhibiting drugs, thereby alleviating cardiac interstitial fibrosis.

[0013] In a third aspect, the present invention provides a small interfering RNA fragment capable of reducing CD248 gene expression, having the following nucleotide sequence:

[0014] SEQ ID NO: 1: Sense(5'-3'):GCCCACAAGACCCUCUGUUAUU;

[0015] SEQ ID NO: 2: Antisense(5'-3'):AAUAACAGAGGUCUUGUGGGC.

[0016] The present invention designs and synthesizes a small interfering RNA sequence targeting the CD248 gene, which can effectively inhibit fibroblast activation and collagen matrix synthesis under TGF-β stimulation.

[0017] In a fourth aspect, the present invention provides the use of the aforementioned small interfering RNA fragment targeting the CD248 gene in the preparation of an anti-fibrosis drug for late ischemic heart disease.

[0018] In a fifth aspect, the present invention provides a monoclonal antibody specific for CD248,

[0019] The amino acid sequence of its heavy chain is shown in SEQ ID NO: 3:

[0020] The amino acid sequence of its light chain is shown in SEQ ID NO: 4:

[0021] The present invention prepares a monoclonal antibody IgG78 targeting CD248, which has good affinity for CD248 protein and can effectively inhibit the activation of fibroblasts, thereby alleviating excessive cardiac fibrosis.

[0022] In a sixth aspect, the present invention provides the use of the above-mentioned specific monoclonal antibody in the preparation of an anti-fibrosis drug for late ischemic heart disease.

[0023] In a seventh aspect, the present invention provides a CAR-T cell targeting CD248, wherein the amino acid sequence of the scFC region is shown in SEQ ID NO: 5:

[0024] The present invention further designs and prepares CD248 CAR-T cells using the Fab segment of monoclonal antibody IgG78. + After injection, CD248 CAR-T cells can infiltrate into the ischemic injury area of ​​the heart and reduce the CD248 + It can reduce the number of cardiac fibroblasts, thereby alleviating cardiac interstitial fibrosis and improving cardiac contractile function.

[0025] In an eighth aspect, the present invention provides the use of the above-mentioned CAR-T cells in the preparation of anti-fibrosis drugs for late ischemic heart disease.

[0026] In a ninth aspect, the present invention provides an anti-fibrosis drug for late ischemic heart disease, comprising the aforementioned RNA fragment or the aforementioned specific monoclonal antibody or the aforementioned CAR-T cell.

[0027] Preferably, the anti-fibrosis drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0028] Preferably, the anti-fibrosis drug is an injectable preparation.

[0029] More preferably, the injection preparation is an injection preparation for intramyocardial injection, intradermal injection, subcutaneous injection, intramuscular injection or intravenous injection.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) This invention innovatively discovered a group of CD248 cells that play a key role in late cardiac fibrosis in ischemic heart disease using single-cell sequencing technology. + Fibroblasts; CD248 protein is specifically and highly expressed only in cardiac fibroblasts in the ischemic damage area, and is expressed at a lower level in other normal tissue cells. Therefore, using CD248 as a target for intervention in late-stage fibrosis of ischemic heart disease can not only effectively inhibit excessive cardiac fibrosis, but also be highly safe.

[0032] (2) The present invention prepares small interfering RNA fragments, monoclonal antibodies and CAR-T cells targeting CD248; wherein, the small interfering RNA fragments can inhibit fibroblast activation and collagen matrix synthesis under TGF-β stimulation; the monoclonal antibody IgG78 has a good affinity for CD248 protein and can effectively inhibit the activation of fibroblasts; and the CAR-T cells have a good infiltration into the ischemic injury area of ​​the heart and can effectively reduce the CD248 in the ischemic area. + Therefore, the monoclonal antibodies and CAR-T cells of the present invention can significantly reduce cardiac interstitial fibrosis and improve cardiac contractile function. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 shows the single-cell sequencing analysis of differential gene expression in fibroblasts at different time phases / regions after myocardial infarction and the expression and distribution characteristics of CD248 in different time phases / regions of myocardial infarction; Figure (a) shows the differential gene expression in fibroblasts in the infarcted area / distal area 3 days and 28 days after myocardial infarction, Figure (b) shows the expression and distribution of CD248 in fibroblasts in the infarcted area / distal area 3 days and 28 days after myocardial infarction, and Figure (c) shows the expression and distribution of CD248 in fibroblasts in the infarcted area / distal area 3 days and 28 days after myocardial infarction. + The proportion of fibroblasts in the infarcted / distal areas 3 and 28 days after myocardial infarction;

[0034] Figure 2 shows that knocking down the CD248 gene inhibits fibroblast transdifferentiation and collagen matrix synthesis under TGF-β stimulation;

[0035] Figure 3 shows that silencing the CD248 gene improves ischemia / reperfusion-induced cardiac contractile dysfunction; Figure (a) shows RT-PCR verification of the knockout efficiency of CD248 gene knockout mice, and Figures (b, c) show cardiac ultrasound monitoring of changes in left ventricular contractile function in wild-type (WT group) and CD248 knockout mice (CD248 KO group) 0-28 days after ischemia / reperfusion injury;

[0036] FIG4 shows that silencing the CD248 gene alleviates ischemia / reperfusion-induced cardiac interstitial fibrosis; FIG4 (a) is a typical image of Sirius red staining of the hearts of mice in each group, and FIG4 (b) is the fibrosis area calculated by Sirius red staining of the hearts of mice in each group;

[0037] Figure 5 shows the affinity of CD248 monoclonal antibody IgG78 to CD248 protein and its function in inhibiting fibroblast activation; Figure (a) shows the affinity of CD248 monoclonal antibody IgG78 to CD248 protein detected by ELISA, and Figure (b) shows that CD248 monoclonal antibody IgG78 inhibits cardiac fibroblast activation stimulated by TGF-β;

[0038] Figure 6 shows that CD248 monoclonal antibody IgG78 improves cardiac contractile dysfunction after ischemia / reperfusion; Figure (a) shows the in vivo dosage and administration process of CD248 monoclonal antibody IgG78, and Figure (b) shows that CD248 monoclonal antibody IgG78 improves cardiac contractile dysfunction 4 weeks after ischemia / reperfusion;

[0039] Figure 7 shows that CD248 monoclonal antibody IgG78 improves cardiac interstitial fibrosis after ischemia / reperfusion; Figure (a) is a typical image of Sirius red staining of the hearts of mice in each group, and Figure (b) is the fibrosis area calculated by Sirius red staining of the hearts of mice in each group;

[0040] Figure 8 shows CD248 CAR-T cells killing CD248 in vitro + fibroblasts;

[0041] Figure 9 shows the infiltration of CD248 CAR-T cells in the ischemic injury area of ​​the heart 24 hours after CD248 CAR-T was reinfused into the tail vein;

[0042] Figure 10 shows that CD248 CAR-T cells injected into the tail vein improve cardiac contractile dysfunction after ischemia / reperfusion. Figure (a) shows the in vivo dosage and administration process of CD248 CAR-T cells, and Figure (b) shows the continuous monitoring of cardiac function in mice after CD248 CAR-T cell injection.

[0043] Figure 11 shows that CD248 CAR-T cells injected into the tail vein improve cardiac interstitial fibrosis after ischemia / reperfusion. Figure (a) shows a typical Sirius red staining image of the hearts of mice in each group, and Figure (b) shows the fibrosis area calculated by Sirius red staining of the hearts of mice in each group.

[0044] Figure 12 shows the evaluation of the toxic side effects of CD248 CAR-T cells on mice after ischemia / reperfusion; Figure (a) shows the detection of inflammatory factors in serum, and Figure (b) shows HE staining to evaluate the morphology and inflammatory cell infiltration of the liver, spleen, and lungs. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the embodiments.

[0046] Overall embodiment

[0047] An intervention target for late fibrosis in ischemic heart disease, wherein the intervention target is CD248 gene, CD248 protein or CD248 + Fibroblasts. CD248 gene, CD248 protein, or CD248 + Fibroblasts can be used as intervention targets to prepare anti-fibrotic drugs for late-stage ischemic heart disease.

[0048] A small interfering RNA fragment capable of reducing CD248 gene expression has the following nucleotide sequence:

[0049] SEQ ID NO: 1: Sense(5'-3'):GCCCACAAGACCCUCUGUUAUU;

[0050] SEQ ID NO: 2: Antisense(5'-3'):AAUAACAGAGGUCUUGUGGGC.

[0051] A monoclonal antibody specific for CD248,

[0052] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 3:

[0053] The amino acid sequence of its light chain is shown in SEQ ID NO: 4:

[0054] MAWTPLLLLLLSHCTGSLSQPVLTQPPSLSASPGASASLTCTLRSDINVGSYRISWYQQKPGSPPQYLLSYKSDSDKQKGSGVPSRFSGSKDASANAGILLISGLQSEDEADYYCMIWHNSAGVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS*. Use of the above monoclonal antibody in the preparation of anti-fibrosis drugs for late ischemic heart disease.

[0055] A CAR-T cell targeting CD248, comprising the amino acid sequence of the scFC region as shown in SEQ ID NO: 5: MASPLTRFLSLNLLLLGESIILGSGEAQPVLTQPPSLSASPGASASLTCTLRSDINVGSYRISWYQQKPGSPPQYLLSYKSDSDKQKGSGVPSRFSGSKDASANAGILLISGLQSEDEADYYCMIWHNSAGVFGGGTKLTVLGGGGSGGGGSGGGGSQVQLQESGGTLVQPGGSLRLSCEASGFTFSNYAMGWVRQTPGKGLEWLSAIRKSGTTPTMRTQSK*. The CAR-T cell is used in the preparation of an anti-fibrotic drug for late-stage ischemic heart disease.

[0056] An anti-fibrotic drug for late-stage ischemic heart disease, comprising a substance that inhibits CD248 gene expression, or the aforementioned monoclonal antibody, or the aforementioned CAR-T cells. Preferably, the drug further comprises a pharmaceutically acceptable carrier and / or excipient. Preferably, the anti-fibrotic drug is an injectable formulation. Further preferably, the injectable formulation is administered via intramyocardial, intradermal, subcutaneous, intramuscular, or intravenous injection.

[0057] Example 1: Single-cell sequencing analysis of the main fibroblast subpopulations involved in cardiac fibrosis in the late stage of myocardial infarction First, to clarify the phenotype, composition and potential functional characteristics of fibroblasts in the microenvironment of normal and post-MI cardiac tissues, we used enzymatic digestion to prepare single-cell suspensions from normal and infarcted and distal cardiac tissues 3 and 28 days after myocardial infarction, and then flow cytometry sorted PDGFRA + DDR2 + Single-cell sequencing of cardiac fibroblasts was performed using the 10× Genomics single-cell sequencing platform for single-cell sequencing and subsequent data processing. After basic sequencing data processing, RNA reads were aligned to the reference genome using STAR software. The proportion of alignments to various genomic regions was calculated to generate unique molecular identifiers (UMIs). Based on the alignment results, the expression level of each gene in each cell was determined.

[0058] In order to find the fibroblast subpopulation that plays a key role in cardiac remodeling in the late stage of myocardial infarction, we analyzed the differentially expressed genes of each group at different times of myocardial infarction (Figure 1A). The analysis found that compared with fibroblasts in other phases / regions, fibroblasts in the infarcted area 28 days after myocardial infarction highly expressed Igfbp6, C4b, C1s1, C3, Trf, Aebp1, Efemp1, Nbl1, Cd248 and Sfrp2. Finally, based on the criteria of "① the target molecule is highly expressed in pathological fibroblasts, but low / no expression in normal tissues and other types of cells; ② to facilitate subsequent CAR-T cell intervention, the target molecule is a membrane protein", we selected CD248 as the target molecule for subsequent research. Further analysis of the proportion of positive cells found that CD248 was expressed in normal (6.55%), infarcted area (1.54%) and distal area (5.33%) 3 days after myocardial infarction, and distal area (3.06%) 28 days after myocardial infarction. + The proportion of CD248 cells in the infarcted area (22.22%) was lower 28 days after myocardial infarction. + The cell ratio increased significantly (Figure 1B, C).

[0059] Example 2: Knockdown of CD248 gene inhibits fibroblast transdifferentiation and collagen matrix synthesis under TGF-β stimulation To explore the effect of knockdown of CD248 gene on fibroblast activation, we designed and synthesized specific siRNA for mouse CD248 (SEQ ID NO: 1; SEQ ID NO: 2), and verified its effect on fibroblast activation under TGF-β stimulation in in vitro cultured neonatal mouse cardiac fibroblasts. The study found that TGF-β (10ng / ml, 48h) treatment can induce neonatal mouse cardiac fibroblast transdifferentiation, which is manifested by high expression of α-SMA and Periostin, and upregulation of interstitial components Fibronectin and Vimentin (Figure 2). Inhibition of CD248 gene expression by siRNA can inhibit fibroblast transdifferentiation and collagen matrix synthesis under TGF-β stimulation, which is manifested by downregulation of α-SMA, Periostin, Fibronectin, and Vimentin (Figure 2).

[0060] Example 3: Silencing the CD248 gene improves ischemia / reperfusion-induced cardiac contractile dysfunction To clarify the key role of CD248 in regulating cardiac function after ischemic injury, we used CRISPR-cas9 technology to construct CD248 gene knockout mice, and with the help of this genotype mouse, we observed the effect of silencing the CD248 gene on cardiac dysfunction after ischemia / reperfusion (I / R). First, RT-PCR detection confirmed the absence of CD248 molecules in CD248 KO mice (Figure 3A). Subsequently, cardiac ultrasound results suggested that the cardiac function of the sham-operated groups of wild-type (WT) and CD248 gene knockout (CD248KO) mice was stably maintained at the baseline level, and knocking out CD248 did not affect the cardiac function of mice (Figures 3B, C). After I / R, WT mice showed a significant decline in cardiac systolic function, as evidenced by a significant decrease in left ventricular shortening (FS%) and ejection fraction (EF%), and a significant increase in left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) (Figure 3B, C). Interestingly, CD248 KO mice showed significant improvement in left ventricular systolic function, as evidenced by a significant increase in EF and FS and a significant decrease in LVESV at 28 days after I / R, while no statistically significant change in LVEDV was observed (Figure 3B, C).

[0061] Example 4: Silencing the CD248 gene reduces ischemia / reperfusion-induced cardiac interstitial fibrosis To further clarify the role of CD248 in cardiac interstitial remodeling after ischemic injury, we used CD248 knockout mice to observe the effect of silencing the CD248 gene on cardiac interstitial remodeling after I / R. We took heart tissue from each group of mice, embedded it in paraffin, and stained it with Sirius red after sectioning. The morphological evaluation of the heart tissue of each group of mice was performed. The results showed that compared with the wild-type mouse (WT) group, CD248 gene deficiency (CD248 KO group) did not significantly affect the structure of the heart and interstitial collagen deposition; 4 weeks after I / R injury, the collagen deposition of the cardiac interstitium of WT mice increased, while CD248 deficiency (CD248 KO+I / R group) could significantly reduce the collagen deposition of the cardiac interstitium of mice after I / R (Figure 4).

[0062] Example 5: Affinity of CD248 monoclonal antibody IgG78 to CD248 protein and its function in inhibiting fibroblast activation

[0063] To further promote the potential clinical translation of CD248, we designed and synthesized a monoclonal antibody, IgG78, targeting mouse CD248 (heavy chain, SEQ ID NO: 3; light chain, SEQ ID NO: 4). First, we used ELISA analysis to demonstrate that IgG78 had strong affinity for mouse CD248 (Figure 5A). Subsequently, Western blot analysis revealed that IgG78 treatment inhibited fibroblast activation induced by TGF-β (10 ng / ml, 48 hours), as evidenced by decreased expression of α-SMA and Periostin (Figure 5B).

[0064] Example 6: CD248 monoclonal antibody IgG78 improves cardiac contractile dysfunction after ischemia / reperfusion. Furthermore, we investigated the effect of CD248 monoclonal antibody IgG78 on cardiac dysfunction after ischemia / reperfusion (I / R) in a mouse model. Following cardiac I / R injury, the Sham+IgG78 and I / R+IgG78 groups received intraperitoneal injections of IgG78 at 30 mg / kg twice weekly, while the Sham+IgG and I / R+IgG groups received the same dose of IgG (Figure 6A). The results of the study suggested that long-term treatment with monoclonal antibody IgG78 improved I / R-induced cardiac systolic dysfunction and left ventricular remodeling in mice, as manifested by a significant increase in left ventricular ejection fraction (EF%) and fractional shortening (FS%) and a significant decrease in left ventricular end of systolic volume (LVESV), while there was no statistically significant change in left ventricular end of diastolic volume (LVEDV) (Figure 6B).

[0065] Example 7: CD248 monoclonal antibody IgG78 improves cardiac interstitial fibrosis after ischemia / reperfusion To observe the collagen deposition in the cardiac interstitium of each group of mice, we performed Sirius red staining on the heart tissues of mice treated with CD248 monoclonal antibody IgG78 and found that 4 weeks after I / R injury, the cardiac interstitial collagen deposition in the I / R+IgG group of mice increased, while monoclonal antibody IgG78 intervention (I / R+IgG78 group) could significantly reduce the collagen deposition in the cardiac interstitium of mice after I / R (Figures 7A, B).

[0066] Example 8: CD248 CAR-T cells kill CD248 in vitro + fibroblasts

[0067] We designed the extracellular recognition region of the CAR using the Fab fragment of the monoclonal antibody IgG78 and synthesized a retrovirus overexpressing the CD248 CAR. Subsequently, we isolated Pan T cells from the spleens of 6-8 week-old C57BL / 6J mice using Pan T magnetic beads. After activation with CD3 / CD28 magnetic beads, these cells were transfected with the CD248 CAR retrovirus to generate mouse CD248 CAR-T cells (scFC region SEQ ID NO: 5).

[0068] First, we used CD248 overexpression lentiviral transfection (MOI = 40, 72h) to induce CD248 + Fibroblasts were then co-cultured with CD248 CAR-T cells and CD248 expression was analyzed by flow cytometry. + The proportion of fibroblasts changed. The results are shown in Figures 8A and 5B. Compared with Pan T cells, CD248 CAR-T cell treatment can greatly reduce the proportion of CD248 + The proportion of fibroblasts (4.73±1.31% in CD248 Overexpression+CAR-T (1:8) versus 32.37±1.71% in CD248 Overexpression) suggests that CD248 CAR-T cells are sensitive to CD248 + Fibroblasts have good killing ability.

[0069] Example 9: Infiltration of CD248 CAR-T cells in the cardiac ischemic injury area 24 hours after CD248 CAR-T cell reinfusion via tail vein

[0070] The important function of CAR is to guide T cells to the location of the target antigen. To test the tropism of CD248 CAR-T cells, we infused 2×10 CFSE-labeled T cells 2 weeks after I / R modeling. 6 Pan-T or CD248 CAR-T cells were injected into the heart, and 24 hours later, T cell accumulation in the heart was observed using a small animal imaging system. As shown in Figure 9, the fluorescence signal in the I / R+CAR-T group was significantly enhanced compared to the I / R+Pan T group, suggesting that CD248 CAR-T cells have a stronger cardiac tropism than Pan T cells, and more T cells are recruited to the heart.

[0071] Example 10: Tail vein infusion of CD248 CAR-T cells improves cardiac systolic dysfunction after ischemia / reperfusion. Furthermore, we investigated the potential therapeutic effects of CD248 CAR-T cells on cardiac interstitial fibrosis and cardiac dysfunction after I / R in a mouse I / R model. After successful I / R model establishment, we infused 1×10 7 CAR-T cells were injected into the mice, and ultrasound was used to dynamically detect changes in cardiac function in each group of mice (Figure 10A). The results showed that CD248 CAR-T cell treatment improved I / R-induced cardiac systolic dysfunction and left ventricular remodeling in mice, as evidenced by a significant increase in EF and FS and a significant decrease in LVESV and LVEDV (Figure 10B).

[0072] Example 11: Tail vein reinfusion of CD248 CAR-T cells improves cardiac interstitial fibrosis after ischemia / reperfusion. To further analyze the effect of CD248 CAR-T cells on cardiac interstitial fibrosis after I / R, we collected heart tissue from each group of mice, embedded it in paraffin, and stained it with Sirius red for morphological evaluation. As shown in Figure 11, compared with the I / R group, CD248 CAR-T cell reinfusion significantly reduced collagen deposition in the cardiac interstitium of mice after I / R.

[0073] Example 12: Evaluation of the toxic and side effects of CD248 CAR-T cells on mice after ischemia / reperfusion To further evaluate the safety of CD248 CAR-T cell therapy, we used CBA to detect the levels of inflammatory factors in the serum of each group of mice, and used HE staining to evaluate the morphology and immune cell infiltration of important organs throughout the body. CBA multi-cytokine detection found that CD248 CAR-T cell infusion did not significantly affect the levels of IL-6, IL-10, MCP-1, IFN-γ and TNF-α in the serum of I / R mice, and caused a significant decrease in the level of IL-12p70 (Figure 12A). HE staining found that compared with the I / R group, CD248 CAR-T cell intervention did not significantly affect the tissue structure of the liver, kidney and lung of I / R mice, and no obvious immune cell infiltration was observed in the above tissues (Figure 12B).

[0074] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0075] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A target for intervention of advanced fibrosis in ischemic heart disease, characterized by: The intervention target is CD248 gene, CD248 protein or CD248 + Fibroblasts. 2.CD248 gene, CD248 protein or CD248 + Application of fibroblasts as intervention targets in the preparation of anti-fibrotic drugs for late ischemic heart disease.

3. An RNA fragment for reducing CD248 gene expression, characterized in that: The nucleotide sequence of Sense (5'-3') is shown in SEQ ID NO: 1, and the nucleotide sequence of Antisense (5'-3') is shown in SEQ ID NO:

2.

4. A specific monoclonal antibody against CD248, characterized in that: The amino acid sequence of its heavy chain is shown in SEQ ID NO:3, and the amino acid sequence of its light chain is shown in SEQ ID NO:

4.

5. A CAR-T cell targeting CD248, characterized in that: The amino acid sequence of its scFC region is shown in SEQ ID NO:

5.

6. Use of the RNA fragment according to claim 3, the specific monoclonal antibody according to claim 4, or the CAR-T cell according to claim 5 in the preparation of an anti-fibrotic drug for late ischemic heart disease.

7. An anti-fibrosis drug for late stage ischemic heart disease, characterized in that: Containing the RNA fragment as claimed in claim 3 or the specific monoclonal antibody as claimed in claim 4 or the CAR-T cell as claimed in claim 5.

8. The anti-fibrosis drug according to claim 7, characterized in that: The anti-fibrosis drug further comprises a pharmaceutically acceptable carrier and / or excipient.

9. The anti-fibrosis drug according to claim 7 or 8, characterized in that: The anti-fibrosis drug is an injection preparation.

10. The anti-fibrosis drug according to claim 9, characterized in that: The injection preparation is an injection preparation for intramyocardial injection, intradermal injection, subcutaneous injection, intramuscular injection or intravenous injection.

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