Biomarker for detecting anti-VEGF (vascular endothelial growth factor) treatment typing of DME (dimethyl ether) and application of biomarker

Abstract

The invention provides a biomarker for detecting anti-VEGF treatment typing of diabetic macular edema and extracellular vesicle protein for judging the severity of diabetic retinopathy, and the protein with remarkable expression difference is screened out by analyzing the expression difference of the extracellular vesicle protein in aqueous humor of a patient. Wherein C5 and CD34 can be used as biomarkers of DME anti-VEGF treatment typing, C5 can be used for judging the severity of diabetic retinopathy (DR), important guidance is provided for DME or DR personalized treatment, and the C5 and CD34 have wide clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of medical testing, specifically relating to extracellular vesicle membrane protein biomarker reagents for detecting anti-VEGF treatment subtypes of diabetic macular edema and their applications. Background Technology

[0002] Diabetic macular edema (DME) is a serious complication that can occur in patients with diabetic retinopathy (DR), and can even lead to blindness. DME is one of the leading causes of vision loss in working-age individuals, with a global prevalence of approximately 6.8% among diabetic patients aged 20-79. Based on relevant epidemiological data, it is estimated that there are currently about 38 million people with diabetic retinopathy in my country, of whom more than 12 million have DME. The visual impairment caused by DME has become a pressing issue that needs to be addressed.

[0003] The pathogenesis of retinal edema (DME) is complex, primarily involving alterations in the retinal vascular barrier function, leading to fluid accumulation in the macular region. This fluid accumulation is closely related to intraocular inflammation and elevated levels of vascular endothelial growth factor (VEGF). VEGF induces angiogenesis and excessive vascular permeability in DME through inflammatory responses; therefore, anti-VEGF therapy is currently one of the most promising treatment options for DME, but it is also expensive. Even with repeated injections of anti-VEGF drugs following standard treatment protocols, some patients still do not respond well to anti-VEGF drugs, failing to observe a reduction in macular edema or improvement in vision; or the initial response is good, but the degree of improvement gradually declines over time, resulting in recurrent macular edema. This type of DME, which does not respond well to repeated intravitreal injections of anti-VEGF drugs, is called refractory DME. Anti-VEGF treatment for refractory DME not only increases the medical burden on patients and society but may also delay treatment, having an incalculable impact on the patient's visual recovery.

[0004] Extracellular vesicles (EVs) are nanoscale extracellular vesicles (40-1000 nm) formed by prokaryotic / eukaryotic cells through a series of regulated processes such as endocytosis-fusion-efflux or by budding. They are widely distributed in bodily fluids such as blood, saliva, urine, breast milk, lymph, and ascites, as well as cell culture supernatants. As signaling messengers, they can deliver various active components, such as proteins / peptides, lipids, mRNAs, and microRNAs, between cells to influence gene expression and functional changes in recipient cells. EVs play an important regulatory role in physiological and pathological processes by participating in intercellular communication, cell proliferation, cell migration, angiogenesis, and immune regulation. Extracellular vesicle membrane proteins are important components of EVs and play a crucial role in the recognition of EVs by target cells, determining the cell targeting of EVs. Therefore, analysis of extracellular vesicle membrane proteins has significant application value in disease diagnosis and treatment.

[0005] Currently, biomarker detection for DME is based on endogenous metabolites in serum (WO2023072261A1). While serum testing is convenient, it involves numerous metabolites that are greatly influenced by the metabolism of various organs throughout the body. Therefore, the specificity of serum biomarkers is often unsatisfactory, leading to misdiagnosis and delays in treatment. There are no reports of detecting substances in EVs in the aqueous humor of patients as biomarkers for diagnosing DME. Therefore, finding a specific and efficient biomarker to assist in DME treatment, especially in anti-VEGF therapy, while reducing treatment costs, has become a pressing issue for clinical diagnosis. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides for the first time extracellular vesicle membrane proteins in the aqueous humor as biomarkers for adjunctive treatment of diabetic macular edema, particularly for detecting anti-VEGF treatment subtypes of diabetic macular edema.

[0007] In addition, the present invention also provides extracellular vesicle membrane protein biomarkers that can be used to determine the severity of diabetic retinopathy (DR).

[0008] Therefore, the present invention provides the following technical solution: In a first aspect, the present invention provides the application of a reagent for detecting the expression level of a biomarker in the preparation of a kit for diagnosing anti-VEGF treatment subtyping of diabetic macular edema.

[0009] In some embodiments, the biomarker is an extracellular vesicle membrane protein.

[0010] In some embodiments, the extracellular vesicle membrane protein is derived from the patient's aqueous humor.

[0011] In some embodiments, the extracellular vesicle membrane protein is C5, CD34, or any combination thereof.

[0012] In some embodiments, the anti-VEGF treatment for diabetic macular edema is classified as either primary DME or refractory DME.

[0013] The term "initial DME" refers to a DME patient who is diagnosed with macular edema by imaging examination when they first seek medical attention for the disease, and who has not previously received any drug treatment for the macular edema, including anti-VEGF drugs, glucocorticoids, or laser treatment. The aforementioned refractory DME refers to DME patients who have received at least 3 intravitreal injections of anti-VEGF drugs or hormone therapy, but whose macular edema persists or recurs based on continuous OCT monitoring.

[0014] In some embodiments, the expression of extracellular vesicle membrane proteins C5 and / or CD34 is significantly different between the primary DME and the refractory DME.

[0015] In some embodiments, the expression of extracellular vesicle membrane proteins C5 and CD34 is significantly downregulated in the patients with primary DME.

[0016] In some embodiments, the expression of extracellular vesicle membrane proteins C5 and CD34 is significantly upregulated in the patients with refractory DME.

[0017] In some implementations, the expression of extracellular vesicle membrane proteins C5 and CD34 is significantly upregulated in patients with refractory DME compared to patients with newly diagnosed DME.

[0018] In some embodiments, the reagent is provided for use in the preparation of a kit for diagnosing primary DME.

[0019] In some embodiments, the reagent is provided for use in the preparation of a kit for diagnosing refractory DME.

[0020] In some embodiments, the detection method involves collecting aqueous humor from a patient and measuring the expression levels of extracellular vesicle membrane proteins C5 and CD34 in the aqueous humor.

[0021] In some embodiments, the reagents used to detect the expression levels of biomarkers are reagents employing immunohistochemistry or EVArray chips.

[0022] The second aspect of this invention provides a reagent for determining biomarkers for anti-VEGF treatment subtypes of diabetic macular edema.

[0023] In some embodiments, the biomarker is extracellular vesicle membrane protein C5, CD34, or any combination thereof.

[0024] In some embodiments, the anti-VEGF treatment for diabetic macular edema is classified as either primary DME or refractory DME.

[0025] In some embodiments, the expression of extracellular vesicle membrane proteins C5 or CD34 differs significantly between patients with primary DME and those with refractory DME.

[0026] In some implementations, the expression of extracellular vesicle membrane proteins C5 and CD34 is significantly upregulated in patients with refractory DME compared to patients with newly diagnosed DME.

[0027] In a third aspect, the present invention provides a biomarker for detecting anti-VEGF treatment subtypes of diabetic macular edema, wherein the biomarker is an extracellular vesicle membrane protein.

[0028] In some embodiments, the extracellular vesicle membrane protein is extracellular vesicle membrane protein C5, CD34, or any combination thereof.

[0029] In some embodiments, the extracellular vesicle membrane protein is derived from the patient's aqueous humor.

[0030] In some embodiments, the anti-VEGF treatment for diabetic macular edema is classified as either primary DME or refractory DME.

[0031] In some embodiments, the expression of extracellular vesicle membrane proteins C5 or CD34 differs significantly between patients with primary DME and those with refractory DME.

[0032] In some implementations, the expression of extracellular vesicle membrane proteins C5 and CD34 is significantly upregulated in patients with refractory DME compared to patients with newly diagnosed DME.

[0033] In a fourth aspect, the present invention provides the use of a reagent for detecting the expression level of a biomarker in the preparation of a kit for assessing the severity of diabetic retinopathy (DR).

[0034] In some embodiments, the biomarker is extracellular vesicle membrane protein C5.

[0035] In some implementations, the biomarker C5 is positively correlated with the severity of diabetic retinopathy (DR).

[0036] In some implementations, the diabetic retinopathy is classified according to the severity of the disease into no obvious retinopathy, non-proliferative diabetic retinopathy (NPDR), and proliferative diabetic retinopathy (PDR).

[0037] In a fifth aspect, the present invention provides a kit for classifying anti-VEGF treatment of diabetic macular edema or assessing the severity of diabetic retinopathy (DR), the kit comprising the above-mentioned reagents for detecting the expression levels of biomarkers.

[0038] The present invention has achieved the following beneficial effects: 1. This invention is the first to use extracellular vesicle proteins as biomarkers for the subtyping of anti-VEGF treatment in diabetic macular edema; 2. This invention analyzes the differences in the expression levels of extracellular vesicle proteins in the aqueous humor of patients, and screens out extracellular vesicle membrane proteins C5 and CD34 with significant expression differences. ROC analysis shows that the above proteins have high diagnostic value. Clinical data correlation analysis shows that VEGFR2 is not significantly correlated with macular foveal thickness.

[0039] 3. The extracellular vesicle protein biomarkers provided by this invention can be accurately used to determine the subtype of diabetic macular edema (DME) treated with anti-VEGF therapy, effectively assisting in the treatment of DME while saving treatment costs and reducing patient suffering. They possess the advantages of high sensitivity and high specificity, and have significant application value.

[0040] 4. The correlation between the differentially expressed proteins and the severity of diabetic retinopathy (DR) was further validated using publicly available databases. These results suggest that C5 can be used to assess the severity of diabetic retinopathy (DR), providing important guidance for personalized treatment of diabetic macular edema or diabetic retinopathy, and possesses broad clinical application prospects. Attached Figure Description

[0041] Figure 1 To identify the expression of differentially expressed proteins in primary and refractory DME, a bar chart was created. (* indicates...) P <0.05); Figure 2 To validate the bar chart of differentially expressed proteins in primary and refractory DME, (* indicates...) P <0.05); Figure 3 ROC analysis for differentially expressed proteins; Figure 4 Correlation analysis of differentially expressed proteins and macular center thickness; Figure 5 Protein-protein interaction network analysis for differentially expressed proteins; Figure 6 Correlation analysis of differentially expressed proteins and the severity of DR. Detailed Implementation

[0042] The present invention will be further described by way of examples, but the present invention is not limited to the following examples.

[0043] Unless otherwise specified, all the main raw materials and equipment used in this invention are commercially available.

[0044] Example 1: Sample collection, processing, and differential protein screening 1. Sample Collection Treatment-naïve ME is defined as follows: When a patient first presents with diabetic macular edema, imaging confirms the presence of macular edema, and the patient has not previously received any drug treatment (including anti-VEGF drugs, corticosteroids, etc.) or laser treatment for this condition. Refractory ME is defined as follows: Despite receiving at least three intravitreal injections of anti-VEGF drugs or corticosteroids (with standard intervals), macular edema persists or recurs, as confirmed by continuous OCT monitoring.

[0045] Inclusion criteria: Diabetic retinopathy macular edema group: (1) 50 years old < age ≤ 70 years old; (2) patients diagnosed with type 2 diabetes; (3) diagnosed with DME based on imaging evidence (FFA / OCT); (4) meeting the definition of "newly diagnosed DME" or "refractory DME".

[0046] Exclusion criteria: (1) Previous diagnosis of ocular complications, including but not limited to: primary open-angle glaucoma, primary angle-closure glaucoma, age-related macular degeneration, high myopia choroidal retinopathy, active uveitis, central serous choroidal retinopathy, etc.; (2) History of ocular surgery or infection: patients with a history of open ocular trauma, previous ocular surgery, ocular infection, or inflammation of the affected eye in the past or during the study; (3) Recent ocular surgery: patients who have undergone cataract or vitrectomy within the past 3 months; (4) Severe systemic diseases: heart disease, cirrhosis, renal failure, malignant tumors, and history of transplantation, etc.; (5) Contraindications to angiography: known allergy to sodium fluorescein or indocyanine green, or other contraindications that prevent the performance of FFA or ICGA examination.

[0047] Based on the above inclusion and exclusion criteria (Yibin Zhao, et.al,Int J Surg. 2025 Sep 10.doi: 10.1097 / JS9.0000000000003453.), the grouping and basic information of the patients in the discovery set are shown in Table 1.

[0048] Table 1. Patient Information and Basic Data from the DME Discovery Group

[0049] Before intravitreal injection of anti-VEGF drugs, all enrolled patients underwent anterior chamber paracentesis using a 1 ml syringe to collect approximately 0.1 ml of aqueous humor sample. The following procedures were strictly followed after sample collection: the sample was immediately transferred to a pre-labeled 1 ml EP tube and then stored at -80°C.

[0050] 2. Sample processing and testing Membrane proteins of extracellular vesicles (EVs) in aqueous humor samples were detected using the EVArray technology platform (Beijing Emicrobiology Technology Co., Ltd., EVArray-SP400). The chip contains antibodies targeting 435 sites, with double-duplicate detection wells for each target site. 100 μL of blocking buffer (3% BSA solution) was first added to each well and incubated at room temperature for 30 minutes, then discarded. Next, 100 μL of diluted aqueous humor sample was added and incubated at room temperature for 60 minutes. After aspirating the reaction solution, the cells were washed three times with 200 μL of 1×PBS. Then, 100 μL of biotinylated detection antibody (anti-human CD9 / CD63 / CD81, LifeSpan BioSciences, USA; diluted 1:1000-1:1500 in 1×PBS) was added, and the cells were incubated at room temperature for 30 minutes. After aspirating the solution, the cells were washed three times. Cy3-labeled streptavidin (SA1010, Invitrogen) was added to each well, and the cells were incubated in the dark for 30 minutes. After aspirating the supernatant, the cells were washed three times with 1×PBS. Finally, fluorescence signals were acquired using a 532nm laser scanner, and the fluorescence intensity values ​​were analyzed using GenePix Pro software. The median mean fluorescence value of the duplicate wells was subtracted from the background value of the PBS control wells to the signal value of the antibody spot, and normalized. The batch effect between the chips was then analyzed, and the corrected value was the absolute expression level. Simultaneously, the geometric mean of CD9, CD63, and CD81 was used as an internal control normalization factor, and the relative expression levels were obtained after correction.

[0051] 3. Results of differential protein analysis of aqueous humor EVs Among the 435 EV proteins tested, significant differences were found in the levels of C5 (P=0.0495, FC=2.711), adiponectin (P=0.040, FC=1.543), AFP (P=0.041, FC=2.084), ANXA5 (P=0.042, FC=2.055), ANXA6 (P=0.046, FC=3.468), APOE (P=0.048, FC=2.563), CA125 (P=0.016, FC=2.600), CD14 (P=0.033, FC=1.804), CD34 (P=0.034, FC=2.552), and CD69 (P=0.001, FC=1.744) between the newly diagnosed DME group and the refractory DME group.

[0052] Example 2: Biomarker Validation 1. Sample Collection Based on the inclusion and exclusion criteria described above in Example 1, the grouping and basic information of the patients in the validation set are shown in Table 2.

[0053] Table 2. Patient information and basic details of the DME validation set.

[0054] 2. The sample collection, processing, and detection methods are the same as in Example 1.

[0055] 3. Verify the protein analysis results of EVs in the collection water. Significant differences were found in the levels of EVs BMP (P=0.016, FC=1.664), C5 (P=0.018, FC=1.885), C5b9 (P=0.002, FC=8.539), RANTES (P=0.023, FC=0.508), CD13 (P=0.034, FC=1.559), CD200 (P=0.009, FC=1.823), and CD34 (P=0.038, FC=1.510) in the newly diagnosed DME group and the refractory DME group in the validation set.

[0056] A comparison of the discovery and validation sets revealed that only EV proteins C5 and CD34 showed significant differences in both sets. Specifically, in the discovery set, C5 (P=0.0495, FC=2.711) and CD34 (P=0.034, FC=2.552) showed significant differences. Figure 1 As shown, compared with newly diagnosed DME patients, C5 and CD34 expression was significantly upregulated in patients with refractory DME, with a statistically significant difference (*). p＜ 0.05 ).

[0057] In the validation set C5 (P=0.018, FC=1.885), CD34 (P=0.038, FC=1.510), as... Figure 2 As shown, compared with newly diagnosed DME patients, the expression of EVs protein C5 and CD34 was significantly upregulated in refractory DME patients, showing a statistically significant difference (*). p＜0.05 Therefore, EVs protein C5 and CD34 have high sensitivity and specificity as biomarkers for identifying primary and refractory DME.

[0058] Example 3: ROC analysis of differentially expressed proteins Receiver operating characteristic (ROC) curve analysis was performed on the two differentially expressed proteins in the validation set. The results showed that the AUC value for the C5 model was 1.000 for both differentially expressed proteins, and the AUC value for the CD34 model was also 1.000. Figure 3 The above results indicate that C5 and CD34 have high diagnostic value in the classification of diabetic macular edema treated with anti-VEGF therapy. (Judgment criterion: an AUC value greater than 0.7 is considered to have some diagnostic value).

[0059] Example 4: Correlation analysis between differentially expressed proteins and CMT To analyze whether differentially expressed proteins are correlated with the severity of macular edema in patients, and thus further demonstrate the role of differentially expressed proteins in DME, a correlation analysis was conducted between two differentially expressed proteins and foveal thickness (CMT). The results showed that differentially expressed proteins C5 and CD34 were not correlated with CMT (P>0.05). Figure 4 ).

[0060] Example 5: Protein-protein interaction network analysis Protein-protein interaction networks were constructed using the STRING database (https: / / string-db.org / ) and Cytoscape (V 3.10.3).

[0061] Protein-protein interaction (PPI) network analysis revealed that two differentially expressed proteins interacted with other proteins (100%). The core proteins included C5 and CD34. Figure 5 ).

[0062] Example 6: Correlation analysis of differentially expressed proteins with the severity of diabetic retinopathy (DR) The proteomics data from aqueous humor biopsies of diabetic patients, non-proliferative diabetic retinopathy (NPDR) patients, and proliferative diabetic retinopathy (PDR) patients were downloaded from publicly available databases (DOI 10.1016 / j.cell.2023.09.012, Julian Wolf et al.) for result validation. Specific sample information is shown in Table 3.

[0063] Table 3 Sample Information from Public Databases

[0064] Correlation analysis of the expression levels of two differentially expressed proteins in the newly diagnosed and refractory DME groups identified in this study with the severity of diabetic retinopathy (DR) was performed in a validation protein database. The results showed that C5 was positively correlated with the severity of DR (P < 0.001), while CD34 was not correlated with the severity of DR (P > 0.05). Figure 6 ).

[0065] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. The application of a reagent for detecting the expression level of a biomarker in the preparation of a kit for diagnosing anti-VEGF treatment subtyping of diabetic macular edema, characterized in that, The biomarker is an extracellular vesicle membrane protein.

2. The application according to claim 1, characterized in that, The extracellular vesicle membrane protein is C5, CD34, or any combination thereof.

3. The application according to any one of claims 1-2, characterized in that, The anti-VEGF treatment for diabetic macular edema is classified as either primary DME or refractory DME.

4. The application according to claim 3, characterized in that, The expression of extracellular vesicle membrane proteins C5 and / or CD34 is significantly different between the primary DME and the refractory DME. Preferably, the expression of extracellular vesicle membrane proteins C5 and CD34 is significantly downregulated in patients with primary DME; or, The expression of extracellular vesicle membrane proteins C5 and CD34 was significantly upregulated in the patients with refractory DME. More preferably, compared with patients with newly diagnosed DME, the expression of extracellular vesicle membrane proteins C5 and CD34 is significantly upregulated in patients with refractory DME.

5. The application according to claim 4, characterized in that, The reagent is used in the preparation of a kit for diagnosing primary DME; or, the reagent is used in the preparation of a kit for diagnosing refractory DME.

6. The application according to any one of claims 1-2, characterized in that, The detection method involves collecting aqueous humor from a patient and measuring the expression levels of extracellular vesicle membrane proteins C5 and CD34 in the aqueous humor; preferably, the measurement is performed using immunohistochemistry or EVArray chip measurement.

7. A reagent for detecting the expression level of the biomarker according to any one of claims 1-6.

8. A biomarker for detecting anti-VEGF treatment subtypes of diabetic macular edema, characterized in that, The biomarkers are extracellular vesicle membrane proteins C5, CD34, or any combination thereof, and the anti-VEGF treatment typing is as described in claim 3.

9. The application of a reagent for detecting the expression level of a biomarker in the preparation of a kit for assessing the severity of diabetic retinopathy (DR), characterized in that, The biomarker is extracellular vesicle membrane protein C5.

10. The application according to claim 9, characterized in that, The biomarker extracellular vesicle membrane protein C5 is positively correlated with the severity of diabetic retinopathy (DR).

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