Marker for judging prognosis of anti-mda5 antibody positive dermatomyositis associated interstitial pneumonia and application thereof

Abstract

This invention provides a biomarker for predicting the prognosis of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia and its application. The biomarker is CX3CR1 in PBMCs. + CD14 + The proportion of monocytes helps in the early identification of high-risk patients, facilitating timely adjustments to treatment plans and preventing disease progression. CX3CR1 in PBMCs... + CD14 + The method for detecting monocyte proportion is mature and rapid, allowing for multiple tests at different stages of the disease to dynamically monitor disease changes. CX3CR1 in PBMCs... + CD14 + The results of monocyte proportion detection are presented in numerical form, which is highly objective and unaffected by the operator's experience. Compared with subjective judgment based on clinical symptoms and signs, CX3CR1 in PBMCs... + CD14 + The proportion of monocytes, as a prognostic marker, can quantitatively assess disease severity and treatment response, providing an objective basis for clinical decision-making.

Description

Technical Field

[0001] This invention relates to the field of disease prognosis assessment, specifically to biomarkers for prognostic assessment of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia and their applications. Background Technology

[0002] MDA5 + DM is a subtype of dermatomyositis with prominent clinicopathological features, primarily characterized by skin ulceration and rapidly progressive interstitial pneumonia. Research data shows that patients with this subtype often develop interstitial pneumonia (MDA5). + The incidence of DM-ILD exceeds 80%, and nearly half of these cases progress to rapidly progressive interstitial lung disease. This type of lung injury responds poorly to conventional immunosuppressive therapy, has a poor clinical prognosis, and carries a mortality risk exceeding 40% within 6 months of diagnosis.

[0003] In this clinical context, accurate prognostic assessment of patients has become crucial for developing appropriate treatment plans. Early identification of MDA5 is essential. + DM-ILD, combined with clinical manifestations and biomarker detection for precise risk stratification, helps to achieve individualized precision treatment: for high-risk patients, intensive or targeted therapy should be initiated in a timely manner to seize the best intervention time; for low-risk patients, adverse reactions caused by overtreatment should be avoided.

[0004] Currently, the main clinical challenge lies in the lack of highly specific and reliable blood prognostic biomarkers. Although some studies have suggested that indicators such as KL-6 and CXCL16 have certain value, their correlation with disease activity, prognosis, and outcome remains controversial, and they have not yet been widely used in clinical practice. Therefore, exploring more novel prognostic biomarkers that can effectively predict the progression and mortality risk of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia has become an urgent clinical need to improve the prognosis of these patients and an important research direction for improving the clinical outcomes of this disease. Summary of the Invention

[0005] The purpose of this invention is to provide a novel biomarker for predicting the prognosis of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of this invention provides a biomarker for predicting the prognosis of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia, wherein the biomarker is CX3CR1 in peripheral blood mononuclear cells (PBMCs). + CD14 + The proportion of monocytes.

[0008] In this invention, CX3CR1 + CD14 + The monocyte ratio refers to CX3CR1 + CD14 + Double-positive cells account for a significant proportion of CD14. + The proportion of monocyte subsets, which is calculated as follows:

[0009] CX3CR1 + CD14 + Monocyte ratio = (CX3CR1) + CD14 + (Number of double-positive cells) / (CD14) + (Total number of monocytes) × 100%, CD14 + The total number of monocytes refers to the number of all CD14-positive monocytes identified by CD14 antibody labeling.

[0010] A second aspect of the present invention provides a method for detecting CX3CR1 in peripheral blood mononuclear cells. + CD14 + The use of reagents or kits for monocyte proportions in the preparation of tools for prognostic assessment of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia.

[0011] In some embodiments, the reagent or kit contains a reagent for detecting CX3CR1 in peripheral blood mononuclear cells. + CD14 + A bispecific antibody composition with a monocyte ratio, the bispecific antibody composition comprising an anti-CX3CR1 antibody labeled with a first fluorescent dye and an anti-CD14 antibody labeled with a second fluorescent dye, wherein the excitation wavelength and emission wavelength of the first fluorescent dye and the second fluorescent dye are different.

[0012] In some specific embodiments, one of the first fluorescent dye and the second fluorescent dye is fluorescein isothiocyanate (FITC) and the other is allophycocyanin (APC).

[0013] In some specific embodiments, the mass ratio of the first fluorescent dye-labeled anti-CX3CR1 antibody to the second fluorescent dye-labeled anti-CD14 antibody is 1:0.4~0.6.

[0014] In some specific embodiments, the anti-CX3CR1 antibody is an anti-human CX3CR1 monoclonal antibody, and the anti-CD14 antibody is an anti-human CD14 monoclonal antibody. In some specific embodiments, the detection of CX3CR1 in peripheral blood mononuclear cells... + CD14 +The reagent or kit for monocyte ratio contains FITC-labeled anti-CX3CR1 antibody and APC-labeled anti-CD14 antibody.

[0015] In some embodiments, the prognostic assessment of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia includes the following steps:

[0016] (1) Peripheral blood samples were collected from patients with positive anti-MDA5 antibody dermatomyositis-associated interstitial pneumonia, and peripheral blood mononuclear cells were isolated.

[0017] (2) CX3CR1 in the PBMCs was detected by cell loss assay. + CD14 + The proportion of monocytes, if CX3CR1 is present in the peripheral blood mononuclear cells + CD14 + If the proportion of monocytes is below the critical value, the prognosis is considered good.

[0018] Further, the critical value is 46.5% to 47.5%, for example, any value between any two critical values ​​of 46.5%, 46.6%, 46.7%, 46.8%, 46.9%, 47.0%, 47.1%, 47.2%, 47.3%, 47.4%, 47.5%, or above.

[0019] Due to the application of the above-mentioned technical solution, the present invention has the following advantages compared with the prior art:

[0020] This invention is the first to propose CX3CR1 in PBMC. + CD14 + The proportion of monocytes is a prognostic marker for anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia. It helps in the early identification of high-risk patients, facilitating timely adjustments to treatment plans and preventing disease progression. CX3CR1 in PBMCs... + CD14 +

[0021] The method for detecting monocyte proportion is mature and rapid, allowing for multiple tests at different stages of the disease to dynamically monitor changes in the condition. CX3CR1 in PBMCs... + CD14 + The results of monocyte proportion detection are presented in numerical form, which is highly objective and unaffected by the operator's experience. Compared with subjective judgment based on clinical symptoms and signs, CX3CR1 in PBMCs... + CD14 + The proportion of monocytes, as a prognostic marker, can quantitatively assess disease severity and treatment response, providing an objective basis for clinical decision-making. Attached Figure Description

[0022] Figure 1 CX3CR1 in PBMCs of patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia and healthy controls + CD19 + B cells, CX3CR1 + CD56 + NK cells, CX3CR1 + CD4 + T cells, CX3CR1 + CD8 + T cells and CX3CR1 + CD14 + Graph showing the proportion of monocytes;

[0023] Figure 2 CX3CR1 in PBMCs of patients with positive anti-MDA5 antibody dermatomyositis-associated interstitial pneumonia (DIP) was compared with those in the survival and death groups and the healthy control group. + CD14 + Graph showing the proportion of monocytes;

[0024] Figure 3 For CX3CR1 in PBMC + CD14 + ROC curves for predicting poor prognosis in patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia: monocyte percentage, peripheral blood plasma CXCL16 level, and peripheral blood plasma KL6 level.

[0025] Figure 4 For CX3CR1 in PBMC + CD14 + KM curve of monocyte percentage and 6-month cumulative survival rate in patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia;

[0026] Figure 5 CX3CR1 in PBMCs of patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia + CD14 + The correlation analysis results between the proportion of monocytes and the titer of anti-MDA5 antibody, an existing clinical risk factor;

[0027] Figure 6 CX3CR1 in PBMCs of patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia + CD14 + The results of the correlation analysis between the proportion of monocytes and the ferritin content, an existing clinical risk factor;

[0028] Figure 7 CX3CR1 in PBMCs of patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia+ CD14 + The correlation analysis results between the percentage of monocytes and the existing clinical risk factor FVC%. Detailed Implementation

[0029] To provide more reliable and specific biomarkers for the prognostic diagnosis of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia, the inventors of this application, through extensive and in-depth research on a large number of clinical cases and healthy controls, discovered that CX3CR1 in PBMCs of patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia... + CD14 + The proportion of monocytes was significantly higher in PBMCs than in healthy controls and other positive cells. Further studies revealed that CX3CR1 cells were present in PBMCs. + CD14 + The proportion of monocytes was significantly higher in patients with poor prognosis, especially those who died, in MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia compared to those with good prognosis. Further systematic analysis confirmed the presence of CX3CR1 in PBMCs. + CD14 + The proportion of monocytes has good predictive value for the prognosis of patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia. CX3CR1 in the patient's PBMCs... + CD14 + The proportion of monocytes was significantly positively correlated with current clinical risk factors such as anti-MDA5 antibody titer and ferritin level, and significantly negatively correlated with the percentage of forced vital capacity (FVC%). Based on this, the technical solution of this invention is proposed, namely, CX3CR1 in PBMCs... + CD14 + The proportion of monocytes serves as a biomarker for prognostic assessment of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia.

[0030] In this invention, monocytes are a type of innate immune cell with immune activity in the immune system. The cell membrane of monocytes has many different markers, primarily surface antigens and surface receptors.

[0031] In this invention, peripheral blood mononuclear cells (PBMCs) refer to a population of cells isolated from peripheral blood that have a single nucleus, mainly including lymphocytes (T cells, B cells, NK cells) and monocytes.

[0032] In this invention, monocytes are a type of innate immune cell with immune activity in the immune system. The cell membrane of a monocyte has many different markers, primarily surface antigens and surface receptors.

[0033] In this invention, CX3CR1 + CD14 + The monocyte ratio refers to CX3CR1 + CD14 + Double-positive cells account for a significant proportion of CD14. + The proportion of monocyte subsets is calculated in flow cytometry analysis as follows:

[0034] CX3CR1 + CD14 + Monocyte ratio = (CX3CR1) + CD14 + (Number of double-positive cells) / (CD14) + (Total number of monocytes) × 100%, CD14 + The total number of monocytes refers to the number of all CD14-positive monocytes identified by CD14 antibody labeling in flow cytometry analysis.

[0035] In this invention, CX3CR1 (C-X3-C motif chemokine receptor 1) is an important member of the chemokine receptor family, belonging to the G protein-coupled receptor (GPCR) family. It is a protein or its polypeptide fragment, mainly expressed on the surface of immune cells such as monocytes, NK cells, and T cells, playing a crucial role in inflammatory responses, immune cell migration, and tissue homeostasis. In this invention, the terms "CX3CR1 protein," "CX3CR1 polypeptide," and "CX3CR1 antigen" are used interchangeably, all referring to CX3CR1 expressed on the surface of mammalian (e.g., human) monocytes.

[0036] In this invention, CD14 is an important immune cell surface molecule, a protein or its polypeptide fragment, mainly expressed on the surface of myeloid cells such as monocytes, macrophages, and neutrophils, and is a key molecule for innate immune recognition and the initiation of inflammatory responses. In this invention, the terms "CD14 protein," "CD14 polypeptide," and "CD14 antigen" are used interchangeably, all referring to CD14 expressed on the surface of mammalian (such as human) monocytes.

[0037] In this invention, the terms "anti-CX3CR1 antibody" and "specific antibody against CX3CR1" are used interchangeably. Similarly, the terms "anti-CD14 antibody" and "specific antibody against CD14" are used interchangeably.

[0038] In this invention, anti-CX3CR1 antibodies mainly refer to polyclonal and monoclonal antibodies, especially monoclonal antibodies, that are specific to human CX3CR1 protein or its polypeptide fragments. "Specificity" means that the antibody can bind to the human CX3CR1 gene product or fragment. Preferably, it refers to antibodies that can bind to the human CX3CR1 gene product or fragment but do not recognize or bind to other unrelated antigen molecules, including modified or unmodified antibodies.

[0039] In this invention, anti-CD14 antibodies mainly refer to polyclonal and monoclonal antibodies, especially monoclonal antibodies, that are specific to human CD14 protein or its polypeptide fragments. "Specificity" means that the antibody can bind to the human CD14 gene product or fragment. Preferably, it refers to antibodies that can bind to the human CD14 gene product or fragment but do not recognize or bind to other unrelated antigen molecules, including modified or unmodified antibodies.

[0040] In this invention, not only complete monoclonal or polyclonal antibodies are included, but also antibody fragments with immune activity, such as Fab' or (Fab)2 fragments, antibody heavy chains, antibody light chains, genetically engineered single-chain Fv molecules or chimeric antibodies, such as antibodies that have mouse antibody binding specificity but still retain the antibody portion derived from humans.

[0041] The anti-CX3CR1 antibody and anti-CD14 antibody of the present invention can be prepared using various techniques known to those skilled in the art. For example, purified human CX3CR1 gene product or its antigenic fragment, or human CD14 gene product or its antigenic fragment, can be administered to animals to induce the production of polyclonal antibodies. Similarly, cells expressing human CX3CR1 protein or its antigenic fragment, or cells expressing human CD14 protein or its antigenic fragment, can be used to immunize animals to produce antibodies. If the present invention uses a monoclonal antibody, the monoclonal antibody can be prepared using hybridoma technology (see Kohler et al., Nature 256: 495-497, 1975; Kohler et al., Eur. J. Immunol. 6: 511-519, 1976; Kohler et al., Eur. J. Immunol. 6: 292-295, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981).

[0042] The antibodies of this invention can be obtained using fragments or functional regions of the human CX3CR1 gene product and the human CD14 gene product through conventional immunoassay techniques. These fragments or functional regions can be prepared using recombinant methods or synthesized using a peptide synthesizer. Antibodies binding to the unmodified form of the human CX3CR1 gene product and antibodies binding to the unmodified form of the human CD14 gene product can be produced by immunizing animals with gene products generated in prokaryotic cells (e.g., E. coli); antibodies binding to post-translational modified forms (such as glycosylated or phosphorylated proteins or peptides) can be obtained by immunizing animals with gene products generated in eukaryotic cells (e.g., yeast or insect cells).

[0043] In this invention, anti-CX3CR1 antibody and anti-CD14 antibody are used in combination to detect monocytes (CD14 and CX3CR1 proteins) on the cell surface in specimens (especially peripheral blood samples). + CX3CR1 + (Monocytes).

[0044] In this invention, a dual antibody is used, and flow cytometry is performed to detect the cells.

[0045] In this invention, a threshold value is preset; if CX3CR1 in PBMC... + CD14 + A monocyte percentage below a critical value is considered a good prognosis; otherwise, a poor prognosis. In this application, "good prognosis" means a low mortality rate and a 6-month survival rate of over 90%, while "poor prognosis" means a high mortality rate and a 6-month survival rate of less than 30%. In a representative example, the critical value is 46.9%, when CX3CR1 in PBMCs... + CD14 + A monocyte percentage of 46.9% or higher indicates poor lung function and a poor prognosis, requiring close monitoring and an escalation of the treatment plan; when CX3CR1 in PBMCs... + CD14 + If the percentage of monocytes is less than 46.9%, it indicates that the patient's lung function is acceptable and the prognosis is good.

[0046] The technical solution of the present invention has the following advantages:

[0047] (1) This invention provides a biomarker for the prognostic diagnosis of dermatomyositis-associated interstitial pneumonia based on flow cytometry and dual antibody detection. The detection method is convenient and rapid, making it easier for patients to accept and providing more and better options for clinical diagnosis and treatment.

[0048] (2) It not only helps to make an early diagnosis and take appropriate treatment measures, but also facilitates dynamic monitoring of the treatment response and disease changes of patients with anti-MDA5 antibody-positive dermatomyositis-related interstitial pneumonia.

[0049] (3) The detection sensitivity and specificity are superior to existing biomarkers KL-6 and CXCL16, and the prognostic results are more accurate.

[0050] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the embodiments provided below are for illustrative purposes only and are not intended to limit the invention unless otherwise stated. Therefore, the invention should not be considered as limited to the following embodiments, but should be understood to include any and all obvious variations as a result of the teachings provided herein.

[0051] It should be understood that modifications that do not materially affect the activity of various embodiments of the present invention may also be covered within the definition of the invention provided herein. Therefore, the following examples are intended to illustrate the invention but not to limit it.

[0052] In this invention, unless otherwise specified, the operating methods involved in the embodiments are all conventional methods in the art. Experimental methods without specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the reagent manufacturer.

[0053] The materials and general methods involved in the embodiments are as follows:

[0054] Unless otherwise specified, all reagents and materials (including antibodies) are commercially available products.

[0055] Peripheral blood samples were obtained from healthy individuals undergoing physical examinations and from hospitalized patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia.

[0056] Evaluation methods

[0057] Lung function assessment and patient risk stratification: Record indicators such as forced vital capacity percentage (FVC%) and carbon monoxide diffusing capacity (DLCO) upon admission.

[0058] Based on the patient's lung function level, patients are divided into Stage I (Stage 1): FVC% ≥ 50%; Stage II (Stage 2): FVC% < 50%, able to complete lung function test; Stage III (Stage 3): respiratory failure or unable to complete lung function test.

[0059] Example 1: CX3CR1 in PBMCs of patients with positive anti-MDA5 antibody dermatomyositis-associated interstitial pneumonia and those who died compared with healthy individuals. + CD14 + Mononuclear cell ratio analysis

[0060] (1) Separation of PBMCs in patients:

[0061] Collect 5 mL of heparin-anticoagulated blood from the patient and mix thoroughly by inverting repeatedly. Dilute the blood sample with an equal volume of PBS buffer (pH 7.5). Slowly pour the diluted blood sample onto the wall of a 4 mL lymphocyte separation medium (Ficoll-Paque™ PLUS). Centrifuge at 1200 g with a slow inversion and descent for 10 min. Aspirate the cells from the turbid portion of the centrifuge tube and transfer it to another centrifuge tube containing PBS buffer (pH 7.5). Centrifuge at 600 g with a rapid inversion and descent for 7 min. Discard the supernatant, resuspend the cells in 200 μL, and perform cell counting. Dilute with PBS buffer (pH 7.5) to a cell count of 5 × 10⁻⁶ cells / mL. 6 Quantity / mL, for later use.

[0062] (2) Preparation of mixed fluorescent antibodies:

[0063] The concentrations of anti-human CD14 monoclonal antibody (purchased from Abcam) and anti-human CX3CR1 monoclonal antibody (purchased from Abcam) were adjusted to 15 mg / mL and 25 mg / mL, respectively. The solutions were placed in an ice bath and magnetically stirred for 10 min, ensuring no foaming occurred during stirring. FITC (purchased from Sigma-Aldrich) fluorescent dye was weighed out at a ratio of 0.01 mg of fluorescent dye per mg of antibody protein for labeling CX3CR1. APC (purchased from Sigma-Aldrich) fluorescent dye was weighed out at a ratio of 0.01 mg of fluorescent dye per mg of antibody protein for labeling CD14. The fluorescent dyes were slowly added to the corresponding antibody protein solutions with stirring, completing this step within 5 min. The mixture was then incubated at 4°C and magnetically stirred for 18 hours. The mixture was then removed, centrifuged at 2500 rpm for 25 min, and dialyzed overnight at 4°C using pH 8.0 buffered saline. The overnight dialyzed mixture was passed through a glucose gel G50 column to remove free fluorescent dye. The labeled fluorescent antibodies were collected for identification. The prepared fluorescently labeled anti-CD14 antibody and fluorescently labeled anti-CX3CR1 antibody were mixed uniformly at a mass ratio of 1:0.5, sodium azide was added, and the mixture was dispensed into 1 mL amber bottles and stored at -20°C for later use.

[0064] (3) CX3CR1 in PBMC + CD14 +Flow cytometry combined detection of monocyte proportion with dual anti-antibody assays:

[0065] Five mL of fresh heparinized anticoagulated blood was drawn from the patient and thoroughly mixed by inverting repeatedly. Personal circulatory media (PBMCs) were extracted from the patient. The extracted PBMCs were then analyzed using a hematology analyzer, focusing on monocyte count. The PBMCs were diluted to a final concentration of 5 × 10⁻⁶ cells / mL. 6 100 μL of diluted PBMCs was pipetted and 10 μL of the prepared mixed antibody was added. The mixture was incubated in the dark for 15 min. Then, 200 μL of PBS buffer (pH 7.5) was added, mixed well, and analyzed by flow cytometry using a standard analytical flow cytometer. CX3CR1 + CD19 + B cells, CX3CR1 + CD56 + NK cells, CX3CR1 + CD4 + T cells, CX3CR1 + CD8 + T-cell detection reference CX3CR1 + CD14 + For mononuclear cells, select the corresponding double antibody mixture.

[0066] Comparing CX3CR1 in PBMCs of healthy controls (from routine physical examinations) and patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia. + CD19 + B cell percentage, CX3CR1 + CD56 + NK cell ratio, CX3CR1 + CD4 + T cell percentage, CX3CR1 + CD8 + T cells and CX3CR1 + CD14 + The percentage of monocytes is shown in the results. Figure 1 .

[0067] Figure 1 The results showed that CX3CR1 was present in PBMCs of patients with MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia. + The proportion of cells was significantly higher than that of healthy controls, with CD14 being the most abundant. + Monocytes were the most prominent feature, specifically CX3CR1 in PBMCs of patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia. + CD14 + The proportion of monocytes was significantly higher than that of the healthy control group.

[0068] Comparing CX3CR1 in PBMCs of patients with positive anti-MDA5 antibody dermatomyositis-associated interstitial pneumonia in survival and death groups with healthy controls + CD14 + The percentage of monocytes is shown in the results. Figure 2 .

[0069] Figure 2 The results showed that CX3CR1 was present in PBMCs of patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia. + CD14 + The proportion of monocytes was significantly higher in the group of patients who died from dermatomyositis-associated interstitial pneumonia and were positive for anti-MDA5 antibody. CX3CR1 was also found in the initial PBMCs of these patients. + CD14 + The proportion of monocytes was significantly higher in the surviving patients.

[0070] CX3CR1 in PBMCs of patients with positive anti-MDA5 antibody dermatomyositis-associated interstitial pneumonia + CD14 + The ROC curves for predicting patient prognosis (mortality within 6 months) by monocyte percentage compared to plasma KL-6 and plasma CXCL16 levels are shown in the figure. Figure 3 .

[0071] Figure 3 The display shows CX3CR1 in PBMC. + CD14 + The proportion of monocytes has a strong predictive value for the prognosis (mortality within 6 months) of patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia, with an area under the curve of 0.9051, which is higher than that of plasma KL-6 level (0.5906) and plasma CXCL16 level (0.6960).

[0072] CX3CR1 in PBMC + CD14 + Kaplan-Meier (KM) curves comparing the monocyte percentage (high monocyte percentage group (≥46.9%) vs. low monocyte percentage group (<46.9%)) with the 6-month cumulative survival of patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia are shown in [link to Kaplan-Meier survival curve]. Figure 4 .

[0073] Figure 4 KM curve analysis shows CX3CR1 in PBMC + CD14 + Patients with high monocyte percentage (≥46.9%, calculated by the Youden index) who were positive for anti-MDA5 antibody had a significantly higher mortality rate (P<0.0001).

[0074] Example 2: CX3CR1 in PBMCs of patients with anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia + CD14 + Correlation analysis of monocyte percentage with existing clinical risk factors

[0075] Simultaneously, the levels of pulmonary function FVC%, peripheral blood ferritin, and MDA5 antibody titers were measured in patients with positive anti-MDA5 antibody dermatomyositis-associated interstitial pneumonia, and compared with CX3CR1 in PBMCs. + CD14 + The proportion of monocytes was compared. CX3CR1 in PBMCs. + CD14 + The correlation analysis results between the percentage of monocytes and the anti-MDA5 antibody titer, a pre-existing clinical risk factor in patients with positive anti-MDA5 antibody dermatomyositis-associated interstitial pneumonia, are shown in the table below. Figure 5 The correlation analysis results with ferritin levels, a clinical risk factor, are shown in [the table below]. Figure 6 The correlation analysis results with the clinical risk factor FVC% are shown in [the table below]. Figure 7 .

[0076] Figures 5-7 The display shows CX3CR1 in PBMC. + CD14 + The proportion of monocytes was significantly positively correlated with the patient's anti-MDA5 antibody titer and ferritin level, and significantly negatively correlated with the lung function FVC% level. It was observed that CX3CR1 was present in PBMCs. + CD14 + The proportion of monocytes has practical clinical significance in predicting the prognosis of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia.

[0077] Based on the above results, CX3CR1 in PBMC + CD14 + The monocyte percentage is an ideal biomarker for prognostic assessment of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia, exhibiting a clear advantage over CXCL16 and KL6. When used as a prognostic marker for anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia, the cutoff value is 46.9%, meaning that when CX3CR1... + CD14 + A monocyte percentage ≥46.9% indicates poor lung function, poor prognosis, and a high risk of death, requiring close monitoring and escalation of the treatment regimen (initiation of intensive or targeted therapy); when CX3CR1 + CD14 + When the percentage of monocytes is <46.9%, it indicates a good prognosis and a low risk of death. The current treatment plan can be continued or the treatment plan can be adjusted to a more conservative one.

[0078] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Detection of CX3CR1 in peripheral blood mononuclear cells + CD14 + The use of reagents or kits for monocyte proportions in the preparation of tools for prognostic assessment of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia, specifically the CX3CR1. + CD14 + The proportion of monocytes is CX3CR1 + CD14 + Double-positive cells account for a significant proportion of CD14. + The proportion of monocyte subsets.

2. The use according to claim 1, characterized in that, The detection of CX3CR1 in peripheral blood mononuclear cells + CD14 + The reagent or kit for monocyte ratio contains a bispecific antibody composition comprising an anti-CX3CR1 antibody labeled with a first fluorescent dye and an anti-CD14 antibody labeled with a second fluorescent dye, wherein the excitation and emission wavelengths of the first and second fluorescent dyes are different.

3. The use according to claim 2, characterized in that, One of the first fluorescent dye and the second fluorescent dye is fluorescein isothiocyanate, and the other is allophycocyanin.

4. The use according to claim 2, characterized in that, The mass ratio of the first fluorescent dye-labeled anti-CX3CR1 antibody to the second fluorescent dye-labeled anti-CD14 antibody is 1:0.4~0.

6.

5. The use according to claim 2, characterized in that, The anti-CX3CR1 antibody is an anti-human CX3CR1 monoclonal antibody, and the anti-CD14 antibody is an anti-human CD14 monoclonal antibody.

6. The use according to claim 1, characterized in that, The detection of CX3CR1 in peripheral blood mononuclear cells + CD14 + The reagents or kits for monocyte proportion contain fluorescein isothiocyanate-labeled anti-CX3CR1 antibody and allophycocyanin-labeled anti-CD14 antibody.

7. The use according to claim 1, characterized in that, The prognostic assessment of anti-MDA5 antibody-positive dermatomyositis-associated interstitial pneumonia includes the following steps: (1) Peripheral blood samples were collected from patients with positive anti-MDA5 antibody dermatomyositis-associated interstitial pneumonia, and peripheral blood mononuclear cells were isolated. (2) Flow cytometry was used to detect CX3CR1 in the peripheral blood mononuclear cells. + CD14 + The proportion of monocytes, if CX3CR1 is present in the peripheral blood mononuclear cells + CD14 + If the proportion of monocytes is below the critical value, the prognosis is considered good.

8. The use according to claim 7, characterized in that, The critical value is 46.5%~47.5%.

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