Improved progressive immune checkpoint inhibitor related myocarditis mouse model as well as construction method and application thereof

Abstract

The invention relates to the field of animal disease models, and discloses an improved progressive immune checkpoint inhibitor related myocarditis mouse model as well as a construction method and application thereof. The construction method of the model comprises the steps that 0.1 mL of a Freund's complete adjuvant mixed solution is subcutaneously injected into a mouse on the 0th day and the 7th day, and the solution contains 0.25 mg of a mouse cardiac troponin I immunogenic peptide fragment; the method is characterized in that a PD-1 inhibitor and a CTLA-4 inhibitor are jointly injected into a mouse in a caudal vein mode at the frequency of one time every two days from the eighth day, the injection dosage of each drug is 5 mg / kg per time, and the injection process is carried out for 5 times in total. According to the application, cardiac inflammation caused by ICIs drugs is induced through combined use of various reagents, and a disease outcome stage of a patient in the real world is perfectly simulated through dose control and injection method improvement, so that a bedding effect is achieved for clinical research and drug test.

Description

Technical Field

[0001] This application relates to the technical field of animal disease models, and more specifically, to a modified mouse model of progressive immune checkpoint inhibitor-associated myocarditis, its construction method, and its application. Background Technology

[0002] Immune checkpoint inhibitors (ICIs), including monoclonal antibodies targeting CTLA-4, PD-1, and PD-L1, have become a revolutionary advancement in cancer treatment by relieving the inhibition of T lymphocytes and enhancing their anti-tumor immunity. However, this overactivation of the immune system also disrupts the inherent balance of immune tolerance, leading to a series of immune-related adverse effects (irAEs). Among the many irAEs, cardiotoxic events, especially ICI-related myocarditis, although relatively rare, are severe and have a high mortality rate, posing a serious threat to patients' quality of life. The clinical manifestations of ICI-induced myocarditis are broad and lack specificity. Mild cases may present as subclinical abnormalities in biomarkers or nonspecific physical symptoms, while severe cases can rapidly progress to fatal heart failure, cardiogenic shock, or cardiac arrest, posing a significant challenge to early diagnosis and timely intervention in clinical practice. A thorough understanding of its pathophysiological mechanisms is an urgent prerequisite for developing effective prevention and treatment strategies.

[0003] Currently, a core bottleneck in this field is the lack of preclinical animal models that can highly mimic the manifestations of human diseases. Existing experimental models, such as traditional autoimmune myocarditis models or PD-1 and CTLA-4 gene knockout models, generally suffer from unstable model success rates, mild myocardial inflammatory infiltration, and localized lesions, making it difficult to reproduce the acute, fulminant course and complex immune cell infiltration characteristics observed in clinical ICIs-related myocarditis. With the widespread use of ICIs in clinical cancer treatment, the number of related myocarditis cases continues to rise. Therefore, constructing a reliable mouse model whose pathogenesis and clinical manifestations are highly consistent with human diseases has become a crucial foundation for advancing its mechanistic research and clinical translation.

[0004] It is worth noting that the acute inflammatory response is only the initial stage of the pathological process of ICIs-related myocarditis. Its long-term outcome and cardiac function prognosis largely depend on the subsequent myocardial repair and remodeling process, in which myocardial fibrosis plays a crucial role. In an ideal repair process, the resolution of inflammation should be accompanied by moderate extracellular matrix deposition to maintain the integrity of the cardiac structure. However, in the specific immune microenvironment of ICIs-related myocarditis, this process is easily disrupted, transforming into pathological fibrosis characterized by persistently high expression of pro-fibrotic factors (such as TGF-β and IL-11) and abnormal activation and proliferation of myofibroblasts. This diffuse or focal myocardial fibrosis is the structural basis for increased myocardial stiffness and progressive deterioration of diastolic and systolic function, and may ultimately evolve into irreversible dilated cardiomyopathy and chronic heart failure. Therefore, future mechanistic studies and model evaluations should not be limited to the acute inflammatory phase, but must also include the long-term outcome of how immune activation drives myocardial fibrosis in the core scope of investigation, so as to provide a comprehensive theoretical basis and experimental platform for developing intervention strategies that can block the entire course of the disease (from acute myocarditis to chronic heart failure). Summary of the Invention

[0005] The purpose of this invention is to address the technical problems of existing experimental models, such as unstable model success rates, mild myocardial inflammatory infiltration, and localized lesions, making it difficult to reproduce the acute, fulminant course and complex immune cell infiltration characteristics observed in clinical ICIs-related myocarditis. Therefore, this application provides a mouse model of immunosuppressive drug-induced myocarditis that conforms to the actual progression of human disease, laying the groundwork for subsequent pathophysiological studies, pharmacological screening studies, disease outcome simulation, and even the development of multi-disease animal models.

[0006] To achieve the above-mentioned objectives, this application adopts the following technical solution: In a first aspect, this application provides a method for constructing a modified mouse model of progressive immune checkpoint inhibitor-associated myocarditis, comprising the following steps: Step 1: On day 0 and day 7, mice were subcutaneously injected with 0.1 mL of Freund's complete adjuvant mixture containing 0.25 mg of mouse cardiac troponin I immunogenic peptide. Step 2: Starting from day 8, mice were injected via tail vein with a combination of PD-1 inhibitor and CTLA-4 inhibitor every 2 days. The injection dose of each drug was 5 mg / kg / time. This injection process was repeated a total of 5 times to obtain the modified mouse model of progressive immune checkpoint inhibitor-related myocarditis.

[0007] Furthermore, the mice used were 6-week-old male BALB / c mice weighing 20-25 g.

[0008] Further, the amino acid sequence of the peptide is HARVDKVDEERYDVEAKVTKNITEIADLTQKIYDLRGKFKRPTLRRVRIS (SEQ ID NO:1).

[0009] Furthermore, the construction method also includes the steps of model phenotypic analysis and identification.

[0010] Furthermore, the model phenotypic analysis and identification include, but are not limited to, histopathological analysis, echocardiographic assessment of cardiac function, and detection of serum myocardial injury markers.

[0011] Secondly, this application provides an improved mouse model of progressive immune checkpoint inhibitor-associated myocarditis constructed by the aforementioned construction method.

[0012] Thirdly, this application provides the application of the modified progressive immune checkpoint inhibitor-associated myocarditis mouse model in studying the pathogenesis of immune checkpoint inhibitor-associated myocarditis.

[0013] Fourthly, this application provides the application of the improved progressive immune checkpoint inhibitor-associated myocarditis mouse model in the study of therapeutic interventions for immune checkpoint inhibitor-associated myocarditis.

[0014] Fifthly, this application provides the application of the improved progressive immune checkpoint inhibitor-associated myocarditis mouse model in the screening of drugs for immune checkpoint inhibitor-associated myocarditis.

[0015] Previous literature has clarified the use of Pdcd1 - / - Ctla4 + / - While mouse models can simulate the immune characteristics of ICIs-related myocarditis in the real world, the rapid progression of myocardial inflammation in mice makes long-term prognostic follow-up and treatment intervention difficult. This invention designs a mouse model of ICIs-related myocarditis that does not rely on gene knockout. It induces cardiac inflammation induced by ICI drugs through the combined use of multiple reagents, employs a more realistic drug injection method, and, through dosage control and improved injection techniques, perfectly simulates the disease progression stages in real-world patients, laying the groundwork for clinical research and drug trials.

[0016] In summary, compared to existing technologies, the mouse model of this application exhibits a high correlation between disease progression and myocarditis severity and the modeling time, a very high probability of membrane formation, and immune cell infiltration covering the entire heart. By improving upon previous drug-induced myocarditis modeling (Chinese patent application number 202110829509.7), this model perfectly simulates the clinical outcomes and survival prognosis of patients with ICIs-related myocarditis in the real world. Attached Figure Description

[0017] Figure 1 The procedure for establishing a mouse model of progressive immune checkpoint inhibitor-associated myocarditis was demonstrated.

[0018] Figure 2 Representative images of myocardial inflammation and fibrosis levels at different stages in mice with progressive immune checkpoint inhibitor-associated myocarditis are presented.

[0019] Figure 3 Representative echocardiographic images of mice at different stages of progressive immune checkpoint inhibitor-associated myocarditis are shown. EF: Ejection Fraction; FS: Fractional Shortening of the left ventricle.

[0020] Figure 4 The cTnT values ​​of mice with progressive immune checkpoint inhibitor-associated myocarditis are shown. Detailed Implementation

[0021] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. It is worth noting that, unless otherwise specified, all materials or instruments used in the embodiments of this application are commercially available.

[0022] like Figure 1 As shown, this application provides a method for constructing an improved mouse model of progressive immune checkpoint inhibitor-associated myocarditis, comprising the following steps: Step 1: On day 0 and day 7, mice were subcutaneously injected with 0.1 mL of Freund's complete adjuvant mixture containing 0.25 mg of mouse cardiac troponin I immunogenic peptide. Step 2: Starting from day 8, mice were injected via tail vein with a combination of PD-1 inhibitor and CTLA-4 inhibitor every 2 days. The injection dose of each drug was 5 mg / kg / time. This injection process was repeated a total of 5 times to obtain the modified mouse model of progressive immune checkpoint inhibitor-related myocarditis.

[0023] Step 3: Model phenotypic analysis and identification.

[0024] During the experiment, male BALB / c mice aged 6 weeks and weighing 20-25 g were selected.

[0025] The amino acid sequence of the peptide in step 1 above is HARVDKVDEERYDVEAKVTKNITEIADLTQKIYDLRGKFKRPTLRRVRIS (SEQ ID NO:1).

[0026] The model phenotypic analysis and identification in step 3 above includes, but is not limited to, histopathological analysis, echocardiographic assessment of cardiac function, and detection of serum myocardial injury markers.

[0027] Based on the above construction method, this application also provides an improved mouse model of progressive immune checkpoint inhibitor-associated myocarditis constructed by the above construction method, and the application of the obtained mouse model in studying the pathogenesis of immune checkpoint inhibitor-associated myocarditis, studying therapeutic interventions for immune checkpoint inhibitor-associated myocarditis, and screening drugs for immune checkpoint inhibitor-associated myocarditis.

[0028] Example This embodiment discloses a modified method for constructing a mouse model of progressive immune checkpoint inhibitor-associated myocarditis, including the following steps: Step 1: Select 6-week-old male BALB / c mice weighing 20-25 g as experimental subjects.

[0029] The core process of model construction includes two phases of continuous immune stimulation. First, on the experimental start day (day 0) and day 7, mice were subcutaneously injected with 0.1 mL of a fully emulsified Freund's Adjuvant (CFA, purchased from Sigma-Aldrich, USA) mixture containing 0.25 mg of a synthetically produced mouse cardiac troponin I (cTnI) immunogenic peptide. The amino acid sequence of the cTnI peptide is HARVDKVDEERYDVEAKVTKNITEIADLTQKIYDLRGKFKRPTLRRVRIS, synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd.

[0030] Step 2: After the establishment of the above-mentioned autoimmune foundation, the immune checkpoint blockade phase begins. Starting one week after the first cTnI immunization (i.e., day 8), mice are injected via tail vein with a combination of PD-1 inhibitor (trade name: InVivoMab anti-mouse PD-1) and CTLA-4 inhibitor (trade name: InVivoMab anti-mouse CTLA-4) every 2 days. The dosage of each drug is 5 mg / kg body weight (diluted with physiological saline) each time, and this process is repeated a total of 5 times.

[0031] Through the synergistic effect of the above-mentioned "self-antigen sensitization" and "PD-1 / CTLA-4 pathway inhibition", a mouse model that can simulate the key pathological features of clinical ICIs-related myocarditis can be successfully induced.

[0032] Step 3, Model Phenotypic Analysis and Identification To systematically evaluate the cardiac phenotype of the constructed mouse model, multiple identification indicators, including histopathological, functional, and serological markers, were integrated. The specific steps are as follows: 1) Histopathological analysis: After euthanizing the mice, heart tissue was taken and routinely fixed, embedded in paraffin and sectioned. Then, hematoxylin-eosin (H&E) staining and Masson staining were performed. Key pathological changes such as myocardial inflammatory cell infiltration, myocardial cell arrangement and structural integrity, and myocardial fibrosis level were observed under an optical microscope.

[0033] 2) Echocardiographic assessment of cardiac function: The Vevo2100 ultra-high resolution small animal ultrasound imaging system was used to perform non-invasive, in vivo testing of left ventricular systolic function in mice. Left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (FS) were used as core indicators to quantitatively assess cardiac function status.

[0034] 3) Detection of serum myocardial injury markers: The concentration of the key biomarker cTnT in mouse peripheral plasma was detected by enzyme-linked immunosorbent assay (ELISA) to objectively reflect the degree of myocardial cell damage.

[0035] 4) Statistical Analysis: Experimental data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and Turkey's test was used for post-hoc tests. All analyses were performed using GraphPad Prism 10.0 software. *p < 0.05 was considered statistically significant.

[0036] Experimental results 1) Mold formation rate Forty mice from the same batch were randomly divided into two groups of 20 each. One group was modeled using the method described in this embodiment. After model phenotypic analysis and identification, 17 mice were successfully modeled, with an overall model success rate of 80-90% (17 / 20). The other group was modeled using the method described in the embodiment of application number 202110829509.7. After model phenotypic analysis and identification, 9 mice were successfully modeled, with an overall model success rate of 40-50% (9 / 20).

[0037] Therefore, the modeling method of this application has a higher modeling success rate compared with the existing method (application number 202110829509.7).

[0038] 2) Histological evidence of acute myocardial inflammation During the acute inflammatory phase, the HE staining results of the heart tissues of mice in each group were as follows: Figure 2 As shown.

[0039] The control group mice had well-organized cardiomyocytes, dense tissue structure, and no significant signs of inflammation.

[0040] Thirty days after the first dose of cTnI induction, mice showed significant myocardial inflammation with slight fibrosis. Numerous inflammatory cell infiltrations were observed in the interventricular septum region, while the morphology and structure of cardiomyocytes remained largely intact.

[0041] Forty days after the first dose of cTnI induction, myocardial inflammation in mice decreased, and abnormal fibrosis was obvious. Inflammatory cell infiltration and fibrosis formation were observed in the interventricular septum region, while the morphology and structure of cardiomyocytes remained largely intact.

[0042] The degree of inflammatory infiltration and the trend of fibrosis were significantly better than those observed in previous drug-induced myocarditis models (application number 202110829509.7). Figure 2 It was observed that during the short-term myocarditis stage on day 30, the control group showed well-organized cardiomyocytes and a compact tissue structure in the interventricular septum region. In the experimental model group, significant focal inflammatory cell infiltration was observed in the interventricular septum region, accompanied by blurred cardiomyocyte boundaries, necrosis, and interstitial edema. Masson staining revealed that, unlike the control group, the experimental model group (on day 30) exhibited partial fibrosis characteristics in the myocardium, primarily occurring at the papillary muscle plane. Immunofluorescence staining showed relatively significant CD3 positive signals in the inflammatory infiltration areas, suggesting pathogenic T cell infiltration of myocardial tissue causing the inflammatory phenotype.

[0043] On day 40, during the long-term myocarditis stage, the infiltration of inflammatory cells in the subepicardial region decreased, myocardial cell boundaries became blurred, fiber arrangement became disordered, and interstitial vacuolation worsened. Typical pathological changes such as nuclear atypia, multinucleation, nuclear pyknosis, and nuclear fragmentation appeared. HE staining revealed a clear fibrotic phenotype. All five mice in this group exhibited these significant pathological changes. Masson staining showed that, compared to the experimental model group (day 30), the experimental model group (day 40) showed strong positive fibrosis features in the myocardium, with large areas of positive signal in the interventricular septum visible in the sections. Immunofluorescence staining also showed significant CD3 positive signals in the inflammatory infiltration areas. These pathological features suggest that the myocardial tissue has transitioned from inflammatory infiltration to a fibrotic recovery stage.

[0044] 3) Serum myocardial injury markers were significantly elevated. Analysis of plasma myocardial enzyme profiles showed that, compared with the control group, combined treatment with cTnI and anti-PD-1 / CTLA-4 induced a sharp and statistically significant increase in plasma cTnT levels in mice (which has been confirmed in the literature as the most sensitive marker of myocardial injury). Figure 4 Regardless of whether it is in the inflammatory damage stage or the fibrotic proliferation stage, this result confirms from a serum biochemical perspective that the model can successfully induce significant cardiomyocyte damage.

[0045] 4) Evolution of chronic heart failure To further investigate the chronic outcome of the model, we assessed cardiac function in mice by echocardiography on day 30 (inflammatory injury phase) and day 40 (fibrotic proliferative phase) after modeling. The results are as follows: Figure 3 As shown, the EF and FS of mice in the cTnI+anti-PD-1 / CTLA-4 combined treatment group were significantly lower than those in the control group, indicating that the model successfully simulated the pathological process of ICIs-related myocarditis evolving from acute inflammation to chronic heart failure.

[0046] in conclusion Based on the comprehensive evidence from histopathology, serum biochemistry, and cardiac function, this embodiment successfully established and validated an experimental mouse model capable of simulating key stages of acute inflammation, fibrotic progression, and even old myocardial injury associated with clinical immune checkpoint inhibitor-related myocarditis. This model provides a reliable experimental tool for further investigation into the pathogenesis of this serious immune-related adverse reaction and for subsequent therapeutic intervention studies.

[0047] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for constructing an improved mouse model of progressive immune checkpoint inhibitor-associated myocarditis, comprising, The method comprises the following steps: Step 1, on day 0 and day 7, subcutaneously inject 0.1 mL of a complete Freund's adjuvant mixed solution containing 0.25 mg of a mouse cardiac troponin I immunogenic peptide into the mouse; Step 2, starting from day 8, inject the mouse with a PD-1 inhibitor combined with a CTLA-4 inhibitor via the tail vein at a frequency of once every 2 days, and the injection dose of each agent is 5 mg / kg / time, and the injection is performed for a total of 5 times, thereby obtaining the improved progressive immune checkpoint inhibitor-related myocarditis mouse model.

2. The construction method according to claim 1, characterized in that, The mouse is a 6-week-old male BALB / c mouse with a body weight of 20-25 g.

3. The construction method of claim 1, wherein, The amino acid sequence of the peptide is shown as SEQ ID NO:

1.

4. The construction method of claim 1, wherein, The construction method further comprises a step of model phenotype analysis and identification.

5. An improved progressive immune checkpoint inhibitor-related myocarditis mouse model constructed by the construction method of any one of claims 1-4.

6. The improved progressive immune checkpoint inhibitor-related myocarditis mouse model of claim 5 is used in the study of the pathogenesis of immune checkpoint inhibitor-related myocarditis.

7. The improved progressive immune checkpoint inhibitor-related myocarditis mouse model of claim 5 is used in the study of therapeutic intervention means for immune checkpoint inhibitor-related myocarditis.

8. The improved progressive immune checkpoint inhibitor-related myocarditis mouse model of claim 5 is used in the drug screening of immune checkpoint inhibitor-related myocarditis.

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