WO2025001335A1 - 一种缺血性心脏病晚期纤维化干预靶点及其应用 - Google Patents

一种缺血性心脏病晚期纤维化干预靶点及其应用 Download PDF

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WO2025001335A1
WO2025001335A1 PCT/CN2024/082680 CN2024082680W WO2025001335A1 WO 2025001335 A1 WO2025001335 A1 WO 2025001335A1 CN 2024082680 W CN2024082680 W CN 2024082680W WO 2025001335 A1 WO2025001335 A1 WO 2025001335A1
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fibrosis
car
cardiac
fibroblasts
heart disease
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胡新央
王建安
李国华
倪骋
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Zhejiang University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Definitions

  • 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.
  • ischemic heart disease In patients with ischemic heart disease, the degree of cardiac fibrosis is an important indicator for predicting poor prognosis, and its severity is associated with increased rates of hospitalization, mortality, and adverse cardiac events.
  • the most common and fatal acute myocardial ischemic event in clinical practice is acute myocardial infarction; with the development of emergency interventional surgery, the diseased coronary arteries of some patients will be opened within a certain period of time, but the hearts of such patients will also suffer from ischemia/reperfusion injury.
  • Fibroblasts are the main matrix cell type of the heart, and they play an important role in maintaining the structure and regulating the function of the heart.
  • cardiac fibroblasts are a highly heterogeneous cell type, and the functions of different subpopulations of fibroblasts under physiological and pathological conditions are different.
  • fibroblasts are activated and transdifferentiated into myofibroblasts after being stimulated by hypoxia, inflammation, and other damage stimuli. They migrate to the site of injury and synthesize and secrete collagen matrix, which promotes the formation of fibrous scars, thereby repairing damaged myocardial tissue and preventing heart rupture.
  • Fibrogenic myofibroblasts can overproduce extracellular matrix in the long term of myocardial infarction, promoting cardiac interstitial fibrosis; while inhibiting myofibroblast function or clearing specific myofibroblasts can lead to cardiac remodeling. Fibroblast subsets are potential targets for fibrosis intervention.
  • CAR-T cell technology in the field of oncology enables T cells to target and kill pathological cells carrying specific antigens, and CAR combines intracellular T cell signals and co-stimulatory molecules with single-chain variable regions of specific antigens.
  • CAR-T cell technology has completely changed the treatment strategy for refractory acute lymphoblastic leukemia and diffuse large B-cell lymphoma.
  • finding intervention targets that are specifically expressed in pathological fibroblasts and low/no expression in other cells and tissues is the key to ensuring its safety and effectiveness.
  • the present invention provides a target for intervention of late fibrosis in ischemic heart disease and its application.
  • the present invention uses single-cell sequencing technology to discover a group of CD248 + fibroblasts that play a key role in late cardiac fibrosis in ischemic heart disease; CD248 protein is only specifically and highly expressed in cardiac fibroblasts in the ischemic injury area, and the expression level in other normal tissue cells is low. Therefore, using CD248 as a target for intervention of late fibrosis in ischemic heart disease can not only effectively inhibit excessive cardiac fibrosis, but also has high safety.
  • the present invention prepares RNA fragments, monoclonal antibodies and CAR-T cells for CD248; among them, small interfering RNA fragments can inhibit fibroblast activation and collagen matrix synthesis under TGF- ⁇ stimulation; monoclonal antibody IgG78 has good affinity for CD248 protein and can effectively inhibit fibroblast activation; CAR-T cells have good infiltration in the ischemic injury area of the heart, and can effectively reduce the number of CD248 + fibroblasts 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.
  • the present invention provides an intervention target for advanced fibrosis in ischemic heart disease, wherein the intervention target is CD248 gene, CD248 protein or CD248 + fibroblasts.
  • 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 + fibroblasts that play a key role in cardiac fibrosis in the late stage of ischemic heart disease; CD248 protein is only specifically highly expressed in activated cardiac fibroblasts, especially in the late stage of ischemic heart disease, which is one of the main factors leading to excessive cardiac fibrosis; and, the expression level of CD248 protein in normal tissue cells is low, so it has high safety as an intervention target.
  • the present invention provides the use of CD248 gene, CD248 protein or CD248 + fibroblasts as intervention targets in the preparation of anti-fibrosis drugs for late ischemic heart disease.
  • drugs can be prepared in a targeted manner that can inhibit CD248 gene expression, inhibit CD248 protein activity, reduce the number of CD248 proteins, or kill CD248 + fibroblasts, thereby alleviating cardiac interstitial fibrosis.
  • the present invention provides a small interfering RNA fragment capable of reducing the expression of the CD248 gene, having the following nucleotide sequence:
  • SEQ ID NO: 1 Sense(5’-3’):GCCCACAAGACCUCUGUUAUU;
  • SEQ ID NO: 2 Antisense(5’-3’):AAUAACAGAGGUCUUGUGGGC.
  • the present invention designs and synthesizes a small interfering RNA sequence targeting the CD248 gene, which can effectively inhibit the activation of fibroblasts and the synthesis of collagen matrix under the stimulation of TGF- ⁇ .
  • the present invention provides the use of the above-mentioned small interfering RNA fragment targeting CD248 gene in the preparation of an anti-fibrosis drug for late ischemic heart disease.
  • the present invention provides a specific monoclonal antibody against CD248,
  • amino acid sequence of its heavy chain is shown in SEQ ID NO: 3:
  • amino acid sequence of its light chain is shown in SEQ ID NO: 4:
  • the present invention prepares a monoclonal antibody IgG78 targeting CD248, which has good affinity to CD248 protein and can effectively inhibit the activation of fibroblasts, thereby alleviating excessive cardiac fibrosis.
  • the present invention provides the above-mentioned specific monoclonal antibody for the preparation of an anti-fibrosis drug for late ischemic heart disease. Application in things.
  • the present invention provides a CAR-T cell targeting CD248, wherein the amino acid sequence of the scFC region is as shown in SEQ ID NO: 5:
  • the present invention further designs and prepares CD248 CAR-T cells using the Fab segment of monoclonal antibody IgG78.
  • CD248 CAR-T cells can kill CD248 + fibroblasts; after injection, CD248 CAR-T cells can infiltrate into the ischemic injury area of the heart well, reduce the number of CD248 + fibroblasts in the ischemic area, thereby reducing cardiac interstitial fibrosis and improving cardiac contractile function.
  • 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.
  • the present invention provides an anti-fibrosis drug for late ischemic heart disease, comprising the above-mentioned RNA fragment or the above-mentioned specific monoclonal antibody or the above-mentioned CAR-T cell.
  • the anti-fibrosis drug further comprises a pharmaceutically acceptable carrier and/or excipient.
  • the anti-fibrosis drug is an injectable preparation.
  • the injection preparation is an injection preparation for intramyocardial injection, intradermal injection, subcutaneous injection, intramuscular injection or intravenous injection.
  • the present invention innovatively uses single-cell sequencing technology to discover a group of CD248 + fibroblasts that play a key role in late-stage cardiac fibrosis in ischemic heart disease; CD248 protein is specifically and highly expressed only in cardiac fibroblasts in the ischemic damage area, and its expression level in other normal tissue cells is relatively low. Therefore, using CD248 as a target for intervention in late-stage fibrosis in ischemic heart disease can not only effectively inhibit excessive cardiac fibrosis, but also has high safety.
  • 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- ⁇ 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 number of CD248 + fibroblasts 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.
  • Figure 1 shows the differential gene expression and The expression and distribution characteristics of CD248 in different phases/regions of myocardial infarction;
  • Figure (a) shows the differential gene expression in fibroblasts in the infarct area/distal area 3 days and 28 days after myocardial infarction
  • Figure (b) shows the expression and distribution of CD248 in fibroblasts in the infarct area/distal area 3 days and 28 days after myocardial infarction
  • Figure (c) shows the proportion of CD248 + fibroblasts in fibroblasts in the infarct area/distal area 3 days and 28 days after myocardial infarction;
  • FIG2 shows that knocking down the CD248 gene inhibits fibroblast transdifferentiation and collagen matrix synthesis under TGF- ⁇ stimulation
  • Figure 3 shows that silencing the CD248 gene improves ischemia/reperfusion-induced cardiac contractile dysfunction;
  • Figure (a) is RT-PCR verification of the knockout efficiency of CD248 gene knockout mice, and
  • Figures (b, c) are cardiac ultrasound monitoring of the changes in left ventricular contractile function of wild-type (WT group) and CD248 knockout mice (CD248 KO group) 0-28 days after ischemia/reperfusion injury;
  • FIG4 shows that silencing the CD248 gene alleviates ischemia/reperfusion-induced cardiac interstitial fibrosis;
  • FIG4 (a) is a typical picture of Sirius red staining of the hearts of mice in each group, and
  • FIG4 (b) is the fibrosis area of the hearts of mice in each group obtained by Sirius red staining;
  • FIG5 shows the affinity of CD248 monoclonal antibody IgG78 to CD248 protein and its function of inhibiting fibroblast activation; wherein, FIG5 (a) shows the affinity of CD248 monoclonal antibody IgG78 to CD248 protein detected by ELISA, and FIG5 (b) shows that CD248 monoclonal antibody IgG78 inhibits cardiac fibroblast activation under TGF- ⁇ stimulation;
  • FIG6 shows that CD248 monoclonal antibody IgG78 improves cardiac contractile dysfunction after ischemia/reperfusion; wherein FIG6 (a) shows the in vivo administration dosage and administration process of CD248 monoclonal antibody IgG78, and FIG6 (b) shows that CD248 monoclonal antibody IgG78 improves cardiac contractile dysfunction 4 weeks after ischemia/reperfusion;
  • FIG. 7 shows that CD248 monoclonal antibody IgG78 improves cardiac interstitial fibrosis after ischemia/reperfusion;
  • FIG. (a) is a typical picture of Sirius red staining of the hearts of mice in each group, and FIG. (b) is the fibrosis area of the hearts of mice in each group obtained by Sirius red staining;
  • Figure 8 shows CD248 CAR-T cells killing CD248 + fibroblasts in vitro
  • Figure 9 shows the infiltration of CD248 CAR-T cells in the cardiac ischemic injury area 24 hours after CD248 CAR-T was reinfused into the tail vein;
  • Figure 10 shows that CD248 CAR-T cells were reinfused into the tail vein to improve cardiac contractile dysfunction after ischemia/reperfusion;
  • Figure (a) shows the in vivo administration dosage and administration process of CD248 CAR-T cells, and Figure (b) shows the continuous monitoring of mouse cardiac function after CD248 CAR-T cells were reinfused;
  • Figure 11 shows that CD248 CAR-T cells were reinfused into the tail vein to improve cardiac interstitial fibrosis after ischemia/reperfusion;
  • Figure (a) is a typical picture of Sirius red staining of the hearts of mice in each group, and
  • Figure (b) is the fibrosis area obtained by Sirius red staining of the hearts of mice in each group;
  • Figure 12 is an evaluation of the toxic and side effects of CD248 CAR-T cells on mice after ischemia/reperfusion;
  • Figure (a) is the detection of inflammatory factors in serum, and
  • Figure (b) is HE staining to evaluate the morphology and inflammatory cell infiltration of the liver, spleen, and lungs.
  • a target for intervention of late fibrosis in ischemic heart disease wherein the target is CD248 gene, CD248 protein or CD248 + fibroblasts.
  • CD248 gene, CD248 protein or CD248 + fibroblasts as intervention targets can be used to prepare anti-fibrosis drugs for late ischemic heart disease.
  • a small interfering RNA fragment capable of reducing CD248 gene expression has the following nucleotide sequence:
  • SEQ ID NO: 1 Sense(5’-3’):GCCCACAAGACCUCUGUUAUU;
  • SEQ ID NO: 2 Antisense(5’-3’):AAUAACAGAGGUCUUGUGGGC.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 3:
  • amino acid sequence of its light chain is shown in SEQ ID NO: 4:
  • an anti-fibrotic drug for late ischemic heart disease comprising a substance that has an inhibitory effect on the expression of the CD248 gene or the above-mentioned monoclonal antibody or the above-mentioned CAR-T cell.
  • it also includes a pharmaceutically acceptable carrier and/or excipient.
  • the anti-fibrotic drug is an injection preparation.
  • the injection preparation is an injection preparation for intramyocardial injection, intradermal injection, subcutaneous injection, intramuscular injection or intravenous injection.
  • Example 1 Single-cell sequencing analysis of the main fibroblast subpopulations involved in cardiac fibrosis in the late stage of myocardial infarction
  • Single-cell sequencing of cardiac fibroblasts and subsequent data processing were performed using the 10 ⁇ Genomics single-cell sequencing platform.
  • RNA reads were aligned to the reference genome using STAR software, and the proportion of alignment to each segment of the genome was counted to obtain specific unique molecular identifier (UMI) data. Based on the alignment results, we obtained the expression level of each gene in each cell.
  • UMI unique molecular identifier
  • 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 revealed that the proportion of CD248 + cells was low in the 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, while the proportion of CD248 + cells in the infarcted area (22.22%) 28 days after myocardial infarction was significantly increased (Figure 1B, C).
  • Example 2 Knockdown of CD248 gene inhibits fibroblast transdifferentiation and collagen matrix synthesis under TGF- ⁇ stimulation
  • SEQ ID NO: 1 SEQ ID NO: 2
  • TGF- ⁇ 10ng/ml, 48h
  • siRNA to inhibit the gene expression of CD248 can inhibit the transdifferentiation of fibroblasts and collagen matrix synthesis under TGF- ⁇ stimulation, which is manifested by down-regulated expression of ⁇ -SMA, Periostin, Fibronectin, and Vimentin ( Figure 2).
  • Example 3 Silencing the CD248 gene improves ischemia/reperfusion-induced cardiac contractile dysfunction
  • CRISPR-cas9 technology to construct CD248 gene knockout mice, and used this genotype mouse to observe the effect of silencing the CD248 gene on cardiac dysfunction after ischemia/reperfusion (I/R).
  • RT-PCR detection confirmed the absence of CD248 molecules in CD248 KO mice ( Figure 3A).
  • Example 4 Silencing the CD248 gene reduces ischemia/reperfusion-induced cardiac interstitial fibrosis
  • CD248 knockout mice 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 the heart tissue of each group of mice, embedded it in paraffin, sectioned it, and then stained it with Sirius red to perform morphological evaluation on the heart tissue of each group of mice.
  • Example 5 Affinity of CD248 monoclonal antibody IgG78 to CD248 protein and its function of inhibiting fibroblast activation
  • Example 6 CD248 monoclonal antibody IgG78 improves cardiac systolic dysfunction after ischemia/reperfusion Furthermore, we clarified the effect of CD248 monoclonal antibody IgG78 on cardiac dysfunction after I/R in a mouse ischemia/reperfusion (I/R) model. After I/R injury in the mouse heart, the Sham+IgG78 and I/R+IgG78 groups were intraperitoneally injected with IgG78 twice a week at 30 mg/kg, while the Sham+IgG and I/R+IgG groups were treated with the same dose of IgG ( Figure 6A).
  • Example 7 CD248 monoclonal antibody IgG78 improves cardiac interstitial fibrosis after ischemia/reperfusion
  • CD248 monoclonal antibody IgG78 improves cardiac interstitial fibrosis after ischemia/reperfusion
  • 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 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 ( Figure 7A, B).
  • Example 8 CD248 CAR-T cells kill CD248 + fibroblasts in vitro
  • FIGs 8A and 5B The results are shown in Figures 8A and 5B.
  • CD248 CAR-T cell treatment can greatly reduce the proportion of CD248 + fibroblasts (4.73 ⁇ 1.31% in CD248 Overexpression + CAR-T (1:8) versus 32.37 ⁇ 1.71% in CD248 Overexpression), suggesting that CD248 CAR-T cells have a good killing ability on CD248 + fibroblasts.
  • Example 9 Infiltration of CD248 CAR-T cells in the ischemic injury area of the heart 24 hours after CD248 CAR-T cell reinfusion into the tail vein
  • Example 10 Tail vein reinfusion of CD248 CAR-T cells improves cardiac systolic dysfunction after ischemia/reperfusion. Furthermore, we observed the potential therapeutic effect of CD248 CAR-T cells on cardiac interstitial fibrosis and cardiac dysfunction after I/R in a mouse I/R model. After the successful establishment of the I/R model, we used tail vein injection to reinfuse 1 ⁇ 10 7 CAR-T cells 7 days and 14 days after I/R, and dynamically detected the changes in cardiac function of each group of mice by ultrasound (Figure 10A).
  • Example 11 Tail vein infusion of CD248 CAR-T cells improves cardiac interstitial fibrosis after ischemia/reperfusion
  • CD248 CAR-T cells improve cardiac interstitial fibrosis after ischemia/reperfusion
  • the results are shown in Figure 11. The results showed that compared with the I/R group, CD248 CAR-T cell infusion significantly reduced collagen deposition in the cardiac interstitium of mice after I/R.
  • Example 12 Evaluation of the toxic and side effects of CD248 CAR-T cells on mice after ischemia/reperfusion
  • CBA CBA to detect the levels of inflammatory factors in the serum of each group of mice
  • 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- ⁇ and TNF- ⁇ 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).

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Abstract

本发明涉及生物医药领域，公开了一种缺血性心脏病晚期纤维化干预靶点及其应用。本发明发现CD248蛋白仅特异高表达于缺血损伤区的心脏成纤维细胞中，在其他正常组织细胞中表达量较低，因此将其作为缺血性心脏病晚期纤维化干预靶点可有效地抑制心脏过度纤维化，安全性高。本发明制备了针对CD248的RNA片段、单克隆抗体和CAR-T细胞；RNA片段可抑制CD248基因表达；单克隆抗体对CD248蛋白具有较好的亲和力，且可有效抑制成纤维细胞的激活；CAR-T细胞对心脏缺血损伤区具有较好的浸润性，可对CD248+成纤维细胞造成杀伤。

Description

一种缺血性心脏病晚期纤维化干预靶点及其应用 技术领域
本发明涉及生物医药领域，尤其涉及一种缺血性心脏病晚期纤维化干预靶点及其应用。
背景技术
在罹患缺血性心脏病的患者中，心脏纤维化程度是预测不良预后的一个重要指标，其严重程度同患者的住院率、死亡率和不良心脏事件的发生率增加相关。临床最为常见、致命的急性心肌缺血事件为急性心肌梗死；而随着急诊介入手术的开展，部分患者的病变冠脉会在一定时间内被开通，但该类患者心脏同样会遭受缺血/再灌注损伤打击。成纤维细胞是心脏主要的基质细胞类型，其在心脏的结构维持和功能调节中发挥重要作用。越来越多的研究表明，心脏成纤维细胞是一种异质性较高的细胞类型，不同亚群的成纤维细胞在生理及病理情况下的功能有所不同。在心肌梗死早期(2-7天)，成纤维细胞受到缺氧、炎症等损伤刺激后激活转分化为肌成纤维细胞，迁移至损伤部位并合成分泌胶原基质，促发纤维疤痕的产生，从而修复受损的心肌组织、防止心脏破裂。但是在心肌梗死晚(10天以后)，促纤维化信号的持续激活导致胶原基质的过度产生，引发心脏僵硬度增加、顺应性下降，并影响心脏电活动，最终带来心力衰竭的恶性后果。因此，在心肌梗死晚期抑制成纤维细胞过度激活具有积极意义。
通过本发明团队的前期研究，我们发现不同亚群的成纤维细胞在心梗不同时期的作用有所不同，但是由于缺乏可靠的生物标志物，我们对成纤维细胞在心脏稳态及心梗后不同时相的具体作用尚缺乏了解，因此尚无法筛选出在心肌梗死晚期心脏重塑中起关键作用的成纤维细胞亚群。另一方面，目前基础及临床研究中开发应用的抗纤维化靶点和策略作用较宽泛，无法针对特定的病理成纤维细胞亚群，导致抗纤维化的治疗效果不佳且可能带来一定的心脏毒性。近年来单细胞测序技术的快速发展为我们认识心脏成纤维细胞异质性、找寻可靠的生物标志物及治疗靶点提供了有力工具。借助单细胞测序解析心梗后不同时相/区域心脏成纤维细胞亚群的动态变化，进而找寻特异在心梗晚期心脏病理重塑中起关键作用的成纤维细胞亚群，以期在不影响早期瘢痕性修复的前提下改善晚期不良间质重塑，对解决心脏纤维化的难题至关重要。
心肌梗死发生后，成纤维细胞会经历转分化、去分化、凋亡或老化等不同改变，且成纤维细胞的不同命运转归紧密参与心梗后心脏重塑的调节。纤维原性肌成纤维细胞可在心梗远期过度产生细胞外基质，促发心脏间质纤维化；而抑制肌成纤维细胞功能或清除特定的肌 成纤维细胞亚群是潜在的纤维化干预手段。
肿瘤领域新近的嵌合抗原受体(Chimeric antigen receptor,CAR)-T细胞技术赋能T细胞靶向识别杀伤携带特定抗原的病理细胞，而CAR是将细胞内T细胞信号和共刺激分子和特定抗原的单链可变区相组合。CAR-T细胞技术的出现彻底改变了难治性急性淋巴细胞白血病和弥漫性大B细胞淋巴瘤的治疗策略。但是，要将CAR-T技术应用于心脏纤维化的治疗，找寻特异表达于病理成纤维细胞，在其他细胞、组织中低/无表达的干预靶点是保证其安全有效的关键。
发明内容
为了解决上述技术问题，本发明提供了一种缺血性心脏病晚期纤维化干预靶点及其应用。首先，本发明借助单细胞测序技术发现了一群在缺血性心脏病晚期心脏纤维化中起关键作用的CD248+成纤维细胞；CD248蛋白仅特异高表达于缺血损伤区的心脏成纤维细胞中，在其他正常组织细胞中表达量较低，因此将CD248作为缺血性心脏病晚期纤维化干预靶点不仅能够有效地抑制心脏过度纤维化，并且安全性高。其次，本发明制备了针对CD248的RNA片段、单克隆抗体和CAR-T细胞；其中，小干扰RNA片段可抑制TGF-β刺激下的成纤维细胞激活和胶原基质合成；单克隆抗体IgG78对CD248蛋白具有较好的亲和力，且可有效抑制成纤维细胞的激活；CAR-T细胞对心脏缺血损伤区具有较好的浸润性，可有效减少缺血区CD248+成纤维细胞数量，因此，本发明单克隆抗体和CAR-T细胞可显著减轻心脏间质纤维化并提高心脏收缩功能。
本发明的具体技术方案为：
第一方面，本发明提供了一种缺血性心脏病晚期纤维化干预靶点，所述干预靶点为CD248基因、CD248蛋白或CD248+成纤维细胞。
如本申请背景技术中所述，在缺血性心脏病晚期(10天以后)，促纤维化信号的持续激活会导致心脏过度纤维化，最终带来心力衰竭的恶性后果。因此，需要在缺血性心脏病晚期对上述纤维化过程进行抑制。通过本发明团队的前期研究，我们发现不同亚群的成纤维细胞在缺血性心脏病不同时期的纤维化程度各不相同。为此，本发明创新性地借助单细胞测序技术来解析不同时相/区域心脏成纤维细胞亚群的动态变化，最终发现了一群在缺血性心脏病晚期心脏纤维化中起关键作用的CD248+成纤维细胞；CD248蛋白仅特异高表达于激活后的心脏成纤维细胞中，尤其在缺血性心脏病晚期的表达量高，是导致心脏过度纤维化的主要因素之一；并且，CD248蛋白在正常的组织细胞中的表达量较低，因此作为干预靶点具有很高的安全性。
第二方面，本发明提供了CD248基因、CD248蛋白或CD248+成纤维细胞作为干预靶点在制备缺血性心脏病晚期抗纤维化药物中的应用。
利用CD248作为干预靶点，可有针对性地制备可抑制CD248基因表达、可抑制CD248蛋白活性、可减少CD248蛋白数量或杀伤CD248+成纤维细胞的药物，从而减轻心脏间质纤维化。
第三方面，本发明提供了一种可降低CD248基因表达的小干扰RNA片段，具有如下核苷酸序列：
SEQ ID NO：1：Sense(5’-3’):GCCCACAAGACCUCUGUUAUU；
SEQ ID NO：2：Antisense(5’-3’):AAUAACAGAGGUCUUGUGGGC。
本发明设计合成了针对CD248基因的小干扰RNA序列，其可有效抑制TGF-β刺激下的成纤维细胞激活和胶原基质合成。
第四方面，本发明提供了上述针对CD248基因的小干扰RNA片段在制备缺血性心脏病晚期抗纤维化药物中的应用。
第五方面，本发明提供了一种针对CD248的特异性单克隆抗体，
其重链的氨基酸序列如SEQ ID NO：3所示：
其轻链的氨基酸序列如SEQ ID NO：4所示：
本发明制备了针对CD248的单克隆抗体IgG78，其对CD248蛋白具有较好的亲和力，且可有效抑制成纤维细胞的激活，因此可减轻心脏过度纤维化。
第六方面，本发明提供了上述特异性单克隆抗体在制备缺血性心脏病晚期抗纤维化药 物中的应用。
第七方面，本发明提供了一种针对CD248的CAR-T细胞，其scFC区的氨基酸序列如SEQ ID NO：5所示：
本发明进一步利用单克隆抗体IgG78的Fab段设计制备了CD248 CAR-T细胞。CD248CAR-T细胞可对CD248+成纤维细胞造成杀伤；经注射后，CD248 CAR-T细胞可很好地浸润到心脏缺血损伤区，减少缺血区CD248+成纤维细胞数量，进而减轻心脏间质纤维化并提高心脏收缩功能。
第八方面，本发明提供了上述CAR-T细胞在制备缺血性心脏病晚期抗纤维化药物中的应用。
第九方面，本发明提供了一种缺血性心脏病晚期抗纤维化药物，含有上述的RNA片段或上述的特异性单克隆抗体或如上述的CAR-T细胞。
作为优选，所述抗纤维化药物还包括药学上可接受的载体和/或赋形剂。
作为优选，所述抗纤维化药物为注射制剂。
进一步优选，所述注射制剂为通过心肌内注射、皮内注射、皮下注射、肌肉注射或静脉注射的注射制剂。
与现有技术对比，本发明的有益效果是：
(1)本发明创新性地借助单细胞测序技术发现了一群在缺血性心脏病晚期心脏纤维化中起关键作用的CD248+成纤维细胞；CD248蛋白仅特异高表达于缺血损伤区的心脏成纤维细胞中，在其他正常的组织细胞中表达量较低，因此将CD248作为缺血性心脏病晚期纤维化干预靶点不仅能够有效地抑制心脏过度纤维化，并且安全性高。
(2)本发明制备了针对CD248的小干扰RNA片段、单克隆抗体和CAR-T细胞；其中，小干扰RNA片段可抑制TGF-β刺激下的成纤维细胞激活和胶原基质合成；单克隆抗体IgG78对CD248蛋白具有较好的亲和力，且可有效抑制成纤维细胞的激活；而CAR-T细胞对心脏缺血损伤区具有较好的浸润性，可有效减少缺血区CD248+成纤维细胞数量，因此，本发明单克隆抗体和CAR-T细胞可显著减轻心脏间质纤维化并提高心脏收缩功能。
附图说明
图1为单细胞测序分析心肌梗死后不同时相/区域成纤维细胞中的差异基因表达及 CD248在心肌梗死不同时相/区域的表达分布特点；其中，图(a)为心肌梗死后3天、28天梗死区/远端区的成纤维细胞中的差异基因表达，图(b)为CD248在心肌梗死后3天、28天梗死区/远端区的成纤维细胞中的表达、分布情况，图(c)为CD248+成纤维细胞在心肌梗死后3天、28天梗死区/远端区的成纤维细胞中的占比；
图2为敲低CD248基因抑制TGF-β刺激下的成纤维细胞转分化及胶原基质合成；
图3为沉默CD248基因改善缺血/再灌注诱导的心脏收缩功能障碍；其中，图(a)为RT-PCR验证CD248基因敲除小鼠的敲除效率，图(b、c)为心脏超声监测野生型(WT组)及CD248敲除小鼠(CD248 KO组)在缺血/再灌注损伤后0-28天的左心室收缩功能改变；
图4为沉默CD248基因减轻缺血/再灌注诱导的心脏间质纤维化；图(a)为各组小鼠心脏天狼星红染色典型图，图(b)为各组小鼠心脏由天狼星红染色统计得到的纤维化面积；
图5为CD248单克隆抗体IgG78与CD248蛋白的亲和力及其抑制成纤维细胞激活的功能；其中，图(a)为ELISA检测CD248单克隆抗体IgG78与CD248蛋白的亲和力，图(b)为CD248单克隆抗体IgG78抑制TGF-β刺激下的心脏成纤维细胞激活；
图6为CD248单克隆抗体IgG78改善缺血/再灌注后心脏收缩功能障碍；其中，图(a)为CD248单克隆抗体IgG78的在体给药剂量及给药过程，图(b)为CD248单克隆抗体IgG78改善缺血/再灌注后4周心脏收缩功能障碍；
图7为CD248单克隆抗体IgG78改善缺血/再灌注后心脏间质纤维化；图(a)为各组小鼠心脏天狼星红染色典型图，图(b)为各组小鼠心脏由天狼星红染色统计得到的纤维化面积；
图8为CD248 CAR-T细胞体外杀伤CD248+成纤维细胞；
图9为尾静脉回输CD248 CAR-T 24小时后心脏缺血损伤区CD248 CAR-T细胞的浸润情况；
图10为尾静脉回输CD248 CAR-T细胞改善缺血/再灌注后心脏收缩功能障碍；其中，图(a)为CD248 CAR-T细胞的在体给药剂量及给药过程，图(b)为CD248CAR-T细胞回输后的小鼠心脏功能连续监测；
图11为尾静脉回输CD248 CAR-T细胞改善缺血/再灌注后心脏间质纤维化；图(a)为各组小鼠心脏天狼星红染色典型图，图(b)为各组小鼠心脏由天狼星红染色统计得到的纤维化面积；
图12为CD248 CAR-T细胞对缺血/再灌注后小鼠的毒副作用评价；其中，图(a)为血清中炎症因子检测，图(b)为HE染色评估肝脏、脾脏、肺脏的形态和炎症细胞浸润情况。
具体实施方式
下面结合实施例对本发明作进一步的描述。
总实施例
一种缺血性心脏病晚期纤维化干预靶点，所述干预靶点为CD248基因、CD248蛋白或CD248+成纤维细胞。CD248基因、CD248蛋白或CD248+成纤维细胞作为干预靶点可用于制备缺血性心脏病晚期抗纤维化药物。
一种可降低CD248基因表达的小干扰RNA片段，具有如下所示的核苷酸序列：
SEQ ID NO：1：Sense(5’-3’):GCCCACAAGACCUCUGUUAUU；
SEQ ID NO：2：Antisense(5’-3’):AAUAACAGAGGUCUUGUGGGC。
一种针对CD248的特异性单克隆抗体，
重链的氨基酸序列如SEQ ID NO：3所示：
其轻链的氨基酸序列如SEQ ID NO：4所示：
MAWTPLLLLLLSHCTGSLSQPVLTQPPSLSASPGASASLTCTLRSDINVGSYRISWYQQKPGSPPQYLLSYKSDSDKQKGSGVPSRFSGSKDASANAGILLISGLQSEDEADYYCMIWHNSAGVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS*。上述单克隆抗体在制备缺血性心脏病晚期抗纤维化药物中的应用。
一种针对CD248的CAR-T细胞，具有如SEQ ID NO：5所示的scFC区的氨基酸序列：MASPLTRFLSLNLLLLGESIILGSGEAQPVLTQPPSLSASPGASASLTCTLRSDINVGSYRISWYQQKPGSPPQYLLSYKSDSDKQKGSGVPSRFSGSKDASANAGILLISGLQSEDEADYYCMIWHNSAGVFGGGTKLTVLGGGGSGGGGSGGGGSQVQLQESGGTLVQPGGSLRLSCEASGFTFSNYAMGWVRQTPGKGLEWLSAIRKSGTTPTMRTQSK*。上述CAR-T细胞在制备缺血性心脏病晚期抗纤维化药物中的应用。
一种缺血性心脏病晚期抗纤维化药物，含有对CD248基因的表达具有抑制作用的物质或上述的单克隆抗体或如上述的CAR-T细胞。作为优选，还包括药学上可接受的载体和/或赋形剂。作为优选，所述抗纤维化药物为注射制剂。进一步优选，所述注射制剂为通过心肌内注射、皮内注射、皮下注射、肌肉注射或静脉注射的注射制剂。
实施例1：单细胞测序分析参与心梗晚期心脏纤维化的主要成纤维细胞亚群首先，为明确成纤维细胞在正常及心肌梗死后心脏组织微环境中的表型、构成和其在疾病中潜在的功能特征，我们利用酶消化法将正常及心肌梗死后3天、28天梗死区、远端区的心脏组织制成单细胞悬液，而后流式分选了PDGFRA+DDR2+成纤维细胞进行单细胞测序。利用10×Genomics单细胞测序平台进行心脏成纤维细胞单细胞测序及后续数据处理。测序数据经基本处理后，用STAR软件，将RNA reads比对到参考基因组，并统计比对到基因组各个区段的比例，得到特定的独特分子标识符(UMI)数据。根据比对结果，我们得到每个细胞每个基因的表达量。
为找寻在心梗晚期心脏重塑中起关键作用的成纤维细胞亚群，我们基于心梗不同时相对每组的差异表达基因进行了分析(图1A)。分析发现，相较其他时相/区域的成纤维细胞，心梗后28天梗死区的成纤维细胞高表达Igfbp6、C4b、C1s1、C3、Trf、Aebp1、Efemp1、Nbl1、Cd248和Sfrp2。最终，基于“①靶分子高表达于病理成纤维细胞中，而在正常组织及其他类型细胞中低/无表达；②为便于后续的CAR-T细胞干预，靶分子是一种胞膜蛋白”的标准，我们选取CD248作为靶分子进行后续研究。进一步针对阳性细胞比例进行分析发现，正常(6.55％)、心梗后3天梗死区(1.54％)和远端区(5.33％)及心梗后28天的远端区(3.06％)CD248+细胞的比例均较低，而心梗后28天梗死区(22.22％)的CD248+细胞比例显著升高(图1B、C)。
实施例2：敲低CD248基因抑制TGF-β刺激下的成纤维细胞转分化及胶原基质合成为探究敲低CD248基因对成纤维细胞激活的影响，我们设计合成了针对小鼠CD248的特异性siRNA(SEQ ID NO：1；SEQ ID NO：2)，并在离体培养的乳小鼠心脏成纤维细胞中验证了其对TGF-β刺激下成纤维细胞激活的影响。研究发现TGF-β(10ng/ml，48h)处理可诱导乳小鼠心脏成纤维细胞转分化，表现为α-SMA和Periostin高表达，且间质成分Fibronectin及Vimentin表达上调(图2)。而采用siRNA抑制CD248的基因表达，可抑制TGF-β刺激下的成纤维细胞转分化和胶原基质合成，表现为α-SMA、Periostin和Fibronectin、Vimentin的表达下调(图2)。
实施例3：沉默CD248基因改善缺血/再灌注诱导的心脏收缩功能障碍为明确CD248在缺血损伤后心脏功能调节中的关键作用，我们利用CRISPR-cas9技术构建了CD248基因敲除小鼠，并借助该基因型小鼠观察了沉默CD248基因对缺血/再灌注(ischemia/reperfusion,I/R)后心功能障碍的影响。首先，RT-PCR检测证实了CD248 KO小鼠中CD248分子的缺失(图3A)。随后，心脏超声结果提示，野生型(WT)和CD248基因 敲除(CD248KO)小鼠的假手术组心脏功能稳定维持在基线水平，敲除CD248并未对小鼠的心脏功能产生影响(图3B、C)。而I/R造模后，WT小鼠的心脏收缩功能出现明显下降，表现为左室缩短分数(Fractional shortening％,FS％)和射血分数(Ejection fraction％,EF％)显著降低，且左心室舒张末期容积(Left ventricular end of diastolic volume,LVEDV)和左心室收缩末期容积(Left ventricular end of systolic volume,LVESV)显著增加(图3B、C)。非常有意思的是，CD248 KO小鼠的左心室收缩功能得到明显改善，表现为I/R 28天时EF、FS的显著上调和LVESV的显著下调，而LVEDV改变无统计学意义(图3B、C)。
实施例4：沉默CD248基因减轻缺血/再灌注诱导的心脏间质纤维化为进一步明确CD248在缺血损伤后心脏间质重塑中的作用，我们借助CD248基因敲除小鼠观察了沉默CD248基因对I/R后心脏间质重塑的影响。我们取各组小鼠心脏组织，经石蜡包埋、切片后行天狼星红染色，对各组小鼠心脏组织进行形态学评价。结果提示，相较野生型小鼠(WT)组，CD248基因缺失(CD248 KO组)并未显著影响心脏的结构及间质胶原沉积；I/R损伤后4周WT小鼠心脏间质的胶原沉积增加，而CD248缺失(CD248 KO+I/R组)可显著减轻I/R后小鼠心脏间质的胶原沉积(图4)。
实施例5：CD248单克隆抗体IgG78与CD248蛋白的亲和力及其抑制成纤维细胞激活的功能
为进一步推动CD248分子的潜在临床转化，我们设计合成了针对小鼠CD248的单克隆抗体IgG78(重链SEQ ID NO：3和轻链SEQ ID NO：4)。首先，我们利用Elisa观察分析发现，单抗IgG78同小鼠CD248蛋白具有较强的亲和力(图5A)。随后，Western-blot分析发现，单抗IgG78处理抑制了TGF-β(10ng/ml,48h)刺激下的成纤维细胞激活，表现为α-SMA和Periostin的表达水平降低(图5B)。
实施例6：CD248单克隆抗体IgG78改善缺血/再灌注后心脏收缩功能障碍更进一步，我们在小鼠缺血/再灌注(ischemia/reperfusion,I/R)模型上明确了CD248单抗IgG78对I/R后心脏功能障碍的影响。小鼠心脏I/R损伤后，Sham+IgG78及I/R+IgG78组按照30mg/kg的方式每周腹腔注射IgG78两次，而Sham+IgG及I/R+IgG组给予相同剂量的IgG处理(图6A)。研究结果提示，长期的单抗IgG78处理改善I/R诱导的小鼠心脏收缩功能障碍和左心室重塑，表现为左心室射血分数(Ejection fraction％,EF％)、缩短分数(Fractional shortening％,FS％)的显著上调和左心室收缩末期容积(Left ventricular end of systolic volume,LVESV)的显著下调，而左心室舒张末期容积(Left ventricular end of diastolic volume,LVEDV)改变无统计学意义(图6B)。
实施例7：CD248单克隆抗体IgG78改善缺血/再灌注后心脏间质纤维化为观察各组小鼠心脏间质的胶原沉积情况，我们对CD248单克隆抗体IgG78治疗后小鼠的心脏组织进行了天狼星红染色，发现I/R损伤后4周I/R+IgG组小鼠的心脏间质胶原沉积增加，而单抗IgG78干预(I/R+IgG78组)可显著减轻I/R后小鼠心脏间质的胶原沉积(图7A、B)。
实施例8：CD248 CAR-T细胞体外杀伤CD248+成纤维细胞
我们利用单克隆抗体IgG78的Fab段设计了CAR的胞外识别区，并在此基础上制备合成了过表达CD248 CAR的逆转录病毒。随后，我们利用Pan T磁珠从6-8周龄的C57BL/6J小鼠脾脏分选了Pan T细胞，给予CD3/CD28磁珠激活后转染CD248 CAR逆转录病毒制备了小鼠CD248 CAR-T细胞(scFC区SEQ ID NO：5)。
首先，我们采用CD248过表达慢病毒转染(MOI＝40，72h)的方法诱导CD248+成纤维细胞，随后将其与CD248 CAR-T细胞共培养，进而流式分析CD248+成纤维细胞比例改变。结果如图8A、5B所示，相较Pan T细胞，CD248 CAR-T细胞处理可大大降低CD248+成纤维细胞的比例(4.73±1.31％in CD248 Overexpression+CAR-T(1:8)versus 32.37±1.71％in CD248 Overexpression)，提示CD248 CAR-T细胞对CD248+成纤维细胞具有不错的杀伤能力。
实施例9：尾静脉回输CD248 CAR-T 24小时后心脏缺血损伤区CD248 CAR-T细胞的浸润情况
CAR的重要功能是引导T细胞到达靶抗原的位置。为检测CD248 CAR-T细胞的趋向性，我们在I/R造模后2周回输CFSE标记的2×106Pan-T或CD248 CAR-T细胞，24h后利用小动物成像仪观察心脏中T细胞的聚集情况。结果如图9所示，相较I/R+Pan T组，I/R+CAR-T组的荧光信号明显增强，提示相较Pan T细胞，CD248 CAR-T细胞的心脏趋向性更强，更多的T细胞会被募集到心脏区域。
实施例10：尾静脉回输CD248 CAR-T细胞改善缺血/再灌注后心脏收缩功能障碍更进一步，我们在小鼠I/R模型上观察了CD248 CAR-T细胞对I/R后心脏间质纤维化及心脏功能障碍的潜在治疗作用。I/R造模成功后，我们采用尾静脉注射的方式于I/R后7天、14天分别回输1×107CAR-T细胞，并超声动态检测各组小鼠心脏功能的变化(图10A)。研究结果提示，CD248 CAR-T细胞处理改善I/R诱导的小鼠心脏收缩功能障碍和左心室重塑，表现为EF、FS的显著上调和LVESV、LVEDV的显著下调(图10B)。
实施例11：尾静脉回输CD248 CAR-T细胞改善缺血/再灌注后心脏间质纤维化为进一步分析CD248 CAR-T细胞对I/R后心脏间质纤维化的影响，我们取各组小鼠心脏组织，经石蜡包埋、切片后行天狼星红染色，对各组小鼠心脏组织进行形态学评价。结果如图11所 示，相较I/R组，CD248 CAR-T细胞回输显著减轻I/R后小鼠心脏间质的胶原沉积。
实施例12：CD248 CAR-T细胞对缺血/再灌注后小鼠的毒副作用评价为进一步评价CD248 CAR-T细胞治疗的安全性，我们采用CBA法检测了各组小鼠血清中的炎症因子水平，并采用HE染色的方法评估了全身各重要脏器的形态和免疫细胞浸润情况。CBA多细胞因子检测发现，CD248 CAR-T细胞回输并未显著影响I/R小鼠血清中IL-6、IL-10、MCP-1、IFN-γ和TNF-α的水平，且导致IL-12p70的水平显著下降(图12A)。HE染色发现，相较I/R组，CD248 CAR-T细胞干预并未显著影响I/R小鼠肝脏、肾脏和肺脏的组织结构，且上述组织中未见明显免疫细胞浸润(图12B)。
本发明中所用原料、设备，若无特别说明，均为本领域的常用原料、设备；本发明中所用方法，若无特别说明，均为本领域的常规方法。
以上所述，仅是本发明的较佳实施例，并非对本发明作任何限制，凡是根据本发明技术实质对以上实施例所作的任何简单修改、变更以及等效变换，均仍属于本发明技术方案的保护范围。

Claims (10)

  1. 一种缺血性心脏病晚期纤维化干预靶点，其特征在于：所述干预靶点为CD248基因、CD248蛋白或CD248+成纤维细胞。
  2. CD248基因、CD248蛋白或CD248+成纤维细胞作为干预靶点在制备缺血性心脏病晚期抗纤维化药物中的应用。
  3. 一种降低CD248基因表达的RNA片段，其特征在于：Sense(5’-3’)的核苷酸序列如SEQ ID NO：1所示，Antisense(5’-3’)的核苷酸序列如SEQ ID NO：2所示。
  4. 一种针对CD248的特异性单克隆抗体，其特征在于：其重链的氨基酸序列如SEQ ID NO：3所示，轻链的氨基酸序列如SEQ ID NO：4所示。
  5. 一种针对CD248的CAR-T细胞，其特征在于：其scFC区的氨基酸序列如SEQ ID NO：5所示。
  6. 如权利要求3所述RNA片段或权利要求4所述特异性单克隆抗体或权利要求5所述CAR-T细胞在制备缺血性心脏病晚期抗纤维化药物中的应用。
  7. 一种缺血性心脏病晚期抗纤维化药物，其特征在于：含有如权利要求3所述的RNA片段或如权利要求4所述的特异性单克隆抗体或如权利要求5所述的CAR-T细胞。
  8. 如权利要求7所述的抗纤维化药物，其特征在于：所述抗纤维化药物还包括药学上可接受的载体和/或赋形剂。
  9. 如权利要求7或8所述的抗纤维化药物，其特征在于：所述抗纤维化药物为注射制剂。
  10. 如权利要求9所述的抗纤维化药物，其特征在于：所述注射制剂为通过心肌内注射、皮内注射、皮下注射、肌肉注射或静脉注射的注射制剂。
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