CD53 as a biomarker for the diagnosis and prognosis of acute pancreatitis, CD53 neutralizing antibodies and their applications

Abstract

The present invention belongs to the field of biomedicine, and in particular relates to CD53 as a biomarker for the diagnosis and prognosis of acute pancreatitis, CD53 neutralizing antibodies and their applications. The present invention's research confirms the key role of CD53 expression levels in acute pancreatitis. It is the first time that CD53 has been found to be a marker for acute pancreatitis and is associated with the prognosis of patients with acute pancreatitis. Therefore, it can be used as a biomarker for the diagnosis and prognosis of acute pancreatitis. The present invention's research confirms that CD53 neutralizing antibodies can significantly reduce inflammatory damage and NETs formation in pancreatic tissue in mice. CD53 is expected to be developed as an effective biomarker for early diagnosis or prognosis of acute pancreatitis, and CD53 neutralizing antibodies can be used as an effective drug for the prevention or treatment of acute pancreatitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to CD53 as a biomarker for the diagnosis and prognosis of acute pancreatitis, CD53 neutralizing antibodies and their applications. Background Technology

[0002] Acute pancreatitis (AP) is an inflammatory disease of the pancreas characterized by pathological activation of digestive enzymes and self-digestion of pancreatic acinar cells. Most AP patients experience mild symptoms and recover spontaneously. Repeated or excessive acinar cell damage, while causing pancreatic necrosis, releases large amounts of pro-inflammatory mediators and inflammatory chemokines, leading to secondary pancreatic necrosis and infection, systemic inflammatory response, and multiple organ failure, potentially progressing to severe acute pancreatitis (SAP), with a mortality rate as high as 20-40%. Dysregulation of the immune system plays a crucial role in the progression and pathogenesis of SAP. Studies have shown that in the early stages of AP, activated immune cells capture microorganisms and release bactericidal enzymes to alleviate pancreatic inflammation; however, if immune cell-related inflammatory responses are not cleared in time, they can exacerbate pancreatic damage and lead to systemic inflammatory responses. Therefore, finding new targets to target immune cell infiltration and the release of inflammatory mediators to alleviate the severity of AP is urgently needed.

[0003] Neutrophils are the most numerous immune cells in the bloodstream and the final effector cells in acute inflammatory responses, playing a major role in clearing extracellular pathogens. During pancreatic tissue damage in acute pancreatitis (AP), neutrophils are the first to migrate from the circulating blood to the site of injury, and activated neutrophils form neutrophil extracellular traps (NETs). NETs play a crucial role in the development of AP, extensively mediating the inflammatory response and exacerbating the severity of AP. The level of NET formation is also positively correlated with the clinical prognosis of AP. Targeting NET formation and its key components can significantly alleviate pancreatic tissue damage and inflammatory response in AP mice, reduce damage to distant organs, and improve mouse survival rate.

[0004] CD53 (also known as MOX44 or TSPAN25) is a member of a new family of molecules with a four-span membrane domain. It belongs to the panleukocyte antigens of the four-span membrane protein superfamily and is widely expressed on leukocytes, including B cells, T cells, monocytes, and granulocytes. Currently, CD53 plays an important role in the immune system, being involved in B cell development, lymphocyte transport, inflammatory diseases, and tumors. Studies have shown that CD53 plays a role in early B cell development and facilitates lymphocyte recirculation by stabilizing L-selectin on the lymphocyte surface. Furthermore, CD53 can affect cell activation by inhibiting α3 integrin, leading to increased cell adhesion and promoting leukocyte adhesion, extravasation, and aggregation on the endothelium at sites of inflammation.

[0005] To date, the function of CD53 in promoting AP through NET release has not been reported, and no existing technology has demonstrated its related preventive and therapeutic role in acute pancreatitis. Therefore, identifying effective biomarkers for diagnosing AP would significantly promote AP-related prevention and treatment. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides CD53 as a biomarker for the diagnosis and prognosis of acute pancreatitis, CD53 neutralizing antibodies, and their applications. This invention confirms the crucial role of CD53 expression levels in acute pancreatitis, and for the first time discovers that CD53 can serve as a biomarker for acute pancreatitis and is correlated with the prognosis of patients with acute pancreatitis; therefore, it can be used as a biomarker for the diagnosis and prognosis of acute pancreatitis.

[0007] The technical solution provided by this invention is as follows:

[0008] This invention provides the application of CD53 as a diagnostic biomarker in the preparation of products for the diagnosis of acute pancreatitis, said products including test strips or test paper strips, detection chips or kits.

[0009] Furthermore, the diagnosis of acute pancreatitis includes the following steps:

[0010] (1) Collect samples from the subjects to be tested and collect control samples;

[0011] (2) Detect and compare the expression levels of CD53 in the test subject samples and control samples;

[0012] If the expression level of CD53 in the sample of the test subject is higher than that in the control sample, the test subject is diagnosed with acute pancreatitis or at risk of having acute pancreatitis.

[0013] This invention provides the application of CD53 as a prognostic biomarker in the preparation of products for predicting the prognosis of acute pancreatitis, said products including test strips or test paper strips, detection chips or reagent kits.

[0014] Furthermore, the prognostic assessment of acute pancreatitis includes the following steps:

[0015] (1) Samples from patients with post-prognosis acute pancreatitis were collected as the test group, and samples from patients with pre-prognosis acute pancreatitis were collected as the control group;

[0016] (2) Detect and compare the expression levels of CD53 in the samples of the test group and the control group;

[0017] If the expression level of CD53 in the test group is lower than that in the control group, the prognosis of the test group is considered to be good.

[0018] Furthermore, the applicable test samples for the prognostic and pre-prognostic acute pancreatitis patients are blood, plasma, or serum derived from peripheral blood.

[0019] Furthermore, the appropriate test samples for the patients with acute pancreatitis are neutrophils or serum from peripheral blood.

[0020] This invention provides the use of a CD53 neutralizing antibody in the preparation of a medicament for the prevention and / or treatment of acute pancreatitis, wherein the CD53 neutralizing antibody Anti-CD53 downregulates the expression level of CD53.

[0021] Furthermore, the drug is a drug that reduces the expression of serum inflammatory factors to alleviate the inflammatory response.

[0022] Furthermore, the drug is a drug that improves pancreatic tissue damage by inhibiting the formation of extracellular traps of neutrophils.

[0023] Furthermore, the drug is a drug that reduces serum amylase and serum lipase levels.

[0024] Beneficial effects

[0025] 1. This invention confirms the crucial role of CD53 expression level in acute pancreatitis. It is the first time that CD53 has been found to serve as a biomarker for acute pancreatitis. This allows for in-depth research into the entire process of acute pancreatitis pathogenesis, enabling early diagnosis of acute pancreatitis and / or measures to prevent its occurrence. The invention has excellent accuracy, specificity, and sensitivity, and is of significant scientific and clinical value.

[0026] 2. This invention possesses complete follow-up data on acute pancreatitis, and has initially established a blood sample bank of acute pancreatitis patients, currently totaling over 200 cases; it has also skillfully constructed an animal model of acute pancreatitis. Experimental verification shows that the diagnostic marker CD53 of this invention is highly expressed in peripheral blood of acute pancreatitis patients and in neutrophils of mouse pancreatic tissue, and is positively correlated with the clinical prognosis of acute pancreatitis patients.

[0027] 3. This invention confirms that CD53 neutralizing antibodies can significantly inhibit inflammatory damage and NET formation in mouse pancreatic tissue.

[0028] Therefore, CD53 is expected to be developed as an effective biomarker for early diagnosis or prognosis of acute pancreatitis, and CD53 neutralizing antibodies can be used to prepare effective drugs for the prevention or treatment of acute pancreatitis. Attached Figure Description

[0029] Figure 1 This study investigates the expression of CD53 in a PMA-induced in vitro NETs model.

[0030] Figure 1 (A) is a schematic diagram of two sets of transcriptome sequencing data analyzed by Venn diagram after 4 hours of PMA (100 nM) stimulation;

[0031] Figure 1 (B) is a schematic diagram of two sets of transcriptome sequencing data analyzed by volcano plot and thermogram analysis after 4 hours of PMA (100 nM) stimulation;

[0032] Figure 1 (C&D) is a schematic diagram showing the results of detecting CD53 protein levels in neutrophils using Western blotting and the corresponding semi-quantitative analysis.

[0033] Figure 1 (E) is a schematic diagram showing the results of flow cytometry analysis of neutrophil proportion and CD53 expression level.

[0034] Figure 1 (F) is a schematic diagram showing the changes in CD53 mRNA levels in neutrophils after stimulation with PMA (100 nM) at different time gradients (1h, 2h, 3h and 4h) by RT-PCR.

[0035] Figure 2 This study investigates the expression of CD53 in peripheral blood neutrophils of patients with acute pancreatitis (AP).

[0036] Figure 2 (A) is a schematic diagram showing the changes in the expression level of CD53 in human peripheral blood neutrophils detected by flow cytometry;

[0037] Figure 2 (BD) is achieved by detecting CD53 in different groups using Graphpad Prism. + A diagram illustrating the percentage of neutrophils;

[0038] Figure 2 (E) is a schematic diagram of the correlation coefficient heatmap analysis of neutrophil CD53 levels and other clinical indicators in AP patients;

[0039] Figure 2 (F) is an analysis of CD53 using ROC curves. + A diagram illustrating the diagnostic value of PMN, PMN, and CRP levels for MODS;

[0040] Figure 3 This study investigates the expression of CD53 in AP mice.

[0041] Figure 3 (A) is a schematic diagram showing the results of CD53 and MPO in the pancreatic tissue of an AP mouse model induced by taurine through immunofluorescence detection;

[0042] Figure 3 (B) is a schematic diagram of the optical density analysis of CitH3 and MPO fluorescence using ImageJ;

[0043] Figure 4 This describes the application of CD53 neutralizing antibody (Anti-CD53) in PMA-induced NETs;

[0044] Figure 4 (A) is a schematic diagram showing the results of flow cytometry analysis of the expression levels of MPO and ROS in neutrophils stimulated with PMA (100 nM) and incubated with different doses of Anti-CD53 (10, 20 and 40 ng / ml).

[0045] Figure 4 (B) is a magnified schematic diagram of the detection of CitH3 and MPO fluorescence in neutrophils by immunofluorescence and scanning electron microscopy, and neutrophils.

[0046] Figure 4 (C) is a schematic diagram of the optical density analysis of CitH3 and MPO fluorescence using ImageJ;

[0047] Figure 4 (D) is a schematic diagram of the results of detecting the levels of IL-6, TNF-α and MCP-1 in the supernatant by ELISA;

[0048] Figure 5 This describes the application of CD53 neutralizing antibody (Anti-CD53) in PMA-induced NETs;

[0049] Figure 5 (A) is a schematic diagram showing the results of detecting the protein level of PAD4 in neutrophils using Western blotting.

[0050] Figure 5 (B) is a histogram of relative protein expression of PAD4 analyzed using ImageJ;

[0051] Figure 6 This describes the application of CD53 neutralizing antibodies (Anti-CD53) in the prevention / treatment of acute pancreatitis.

[0052] Figure 6 (A) is a schematic diagram of detecting the pancreas of AP mice by HE staining and immunofluorescence staining;

[0053] Figure 6 (B) is a schematic diagram showing the results of serum amylase and lipase levels detected by an enzyme-linked immunosorbent assay (ELISA) reader;

[0054] Figure 6 (C) is a schematic diagram of the optical density analysis of CitH3 and MPO fluorescence using ImageJ;

[0055] Figure 6 (D) is a schematic diagram of pathological scoring of pancreatic tissue using Graphpad Prism;

[0056] Figure 6 (E) is a schematic diagram of the results of detecting the levels of IL-6, TNF-α and MCP-1 in the supernatant by ELISA. Detailed Implementation

[0057] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The provided embodiments are only for illustrating the method of the present invention and are not intended to limit the rest of the contents disclosed in the present invention in any way.

[0058] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods. The reagents and materials used in the embodiments are all commercially available. All quantitative experiments involved in the embodiments were performed in at least three replicates, and the results were averaged.

[0059] The C57BL / 6 mice used were purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd.

[0060] The Caerulein used was purchased from NJPeptide, catalog number 22-09-26.

[0061] The phorbol ester (PMA) used was purchased from MCE, catalog number HY-18739.

[0062] The CD53 neutralizing antibody (Anti-CD53) used was purchased from Biolend, catalog number 124701.

[0063] Example 1: Study on the expression of CD53 in a PMA-induced in vitro NETs model

[0064] PMA-induced mouse bone marrow-derived neutrophil (NET) model.

[0065] Neutrophils were extracted from the bone marrow of male C57BL / 6J mice. The freshly isolated bone marrow neutrophils were then processed at a concentration of 1×10⁻⁶ cells / mL. 7 / ml was seeded into 12-well plates and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2 for 30 minutes. Then, 100 nM PMA was administered to establish an in vitro NETs model, while the control group received an equal volume of PBS. Four hours after modeling, cells were collected, and mRNA was extracted from mouse bone marrow neutrophils before and after PMA induction for RNA-Seq analysis. To reduce batch-to-batch variability, we jointly analyzed the RNA-Seq databases of two batches of NETs models from our research group. We found 16,118 overlapping differentially expressed genes between the two groups, and further analysis revealed significant changes in various cell surface molecules. Combining P-value and Log2FoldChange changes, we used volcano plots and heatmaps to ultimately screen and found that CD53 significantly increased after PMA-induced NETs formation (…). Figure 1 (AB). To further verify the changes in CD53 expression during NET formation, we extracted the protein for Western blotting analysis, flow cytometry analysis, and RT-PCR experiments. Figure 1 As shown in (CD), CD53 protein levels significantly increased 4 hours after PMA modeling. Mouse bone marrow-derived neutrophils were stimulated with PMA (100 nM) for 4 hours, followed by staining for flow cytometry analysis. Figure 1 As shown in (E), bone marrow neutrophils (CD45.2) + CD11b + Ly6G + Representative flow cytometry gating settings are used. Representative flow cytometry plots and bar graphs depicting neutrophil proportions and CD53 expression levels are presented. This shows significantly high CD53 expression in the NET network structure. Figure 1 As shown in (F), RT-PCR analysis of stimulation with PMA (100 nM) at different time gradients (1 h, 2 h, 3 h, and 4 h) showed a significant increase in CD53 mRNA levels. The results indicate that PMA-induced CD53 expression levels were significantly upregulated in NETs compared to the control group.

[0066] Example 2: Study on the expression of CD53 in peripheral blood neutrophils of AP patients.

[0067] Neutrophils were isolated from blood samples collected from patients with acute pancreatitis and healthy controls at the Affiliated Hospital of Yangzhou University.

[0068] Anticoagulated blood samples were collected from patients with acute pancreatitis (AP) and healthy controls at the Affiliated Hospital of Yangzhou University. Neutrophils were isolated using a separation kit, and the extracted neutrophils were analyzed by flow cytometry. Figure 2 As shown in (A), the CD53 level of peripheral blood neutrophils in AP patients was significantly increased compared to the healthy control group. AP patients were divided into the Non-MODS AP group and the MODS AP group according to the modified Marshall scoring system. Based on the presence or absence of local complications or systemic inflammatory response, AP patients were further divided into the Non-LC group and the LC group, and the Non-SIRS group and the SIRS group. Figure 2 As shown in (BD), we found that the expression level of CD53 in peripheral blood neutrophils of AP patients with poor prognosis was significantly higher than that in the control group, and this expression was correlated with disease severity. Figure 2 As shown in (E), variable correlation heatmap analysis revealed a positive correlation between CD53 levels and C-reactive protein (CRP) levels in peripheral blood neutrophils of AP patients. Figure 2 As shown in (F), we found CD53 + Neutrophils are more accurate and sensitive in predicting AP patients who progress to MODS. Therefore, CD53 + Neutrophils have diagnostic value for MODS. Results showed that CD53 expression levels were significantly upregulated in AP patients and correlated with disease severity.

[0069] Example 3: Study on CD53 expression in AP mice

[0070] A mouse model of acute pancreatitis induced by taurine.

[0071] Experimental animals: 6-8 week old male C57 / BL6J mice (average weight 25g).

[0072] An acute pancreatitis model was established in mice by intraperitoneal injection of spirulinaxin (100 μg / kg, 1 hour apart, 10 injections). Control mice received a strict PBS-controlled intraperitoneal injection. Twelve hours after the first spirulinaxin injection, mice were anesthetized and sacrificed using Zolteil (Virbac). Pancreatic tissue was harvested, fixed in 4% paraformaldehyde for 48 hours, embedded in paraffin blocks, sectioned, and then subjected to immunofluorescence staining. Figure 3 As shown in (AB), immunofluorescence detection revealed a significant increase in the co-localization of CD53 and MPO in the damaged pancreatic tissue of the AP group compared to the control group, suggesting that CD53 is primarily highly expressed in neutrophils after AP. The results indicate that CD53 is significantly highly expressed in the pancreatic tissue of AP mice.

[0073] Example 4: Application of CD53 neutralizing antibody (Anti-CD53) in PMA-induced NETs

[0074] PMA-induced mouse bone marrow-derived neutrophil (NET) model.

[0075] Neutrophils were extracted from the bone marrow of male C57BL / 6J mice. Freshly isolated bone marrow neutrophils were seeded at 1×10⁷ / ml into 12-well plates and cultured at 37°C in a 5% CO₂ incubator on RPMI 1640 medium containing 10% fetal bovine serum (FBS) for 30 minutes. The cells were then divided into the following groups:

[0076] Control group: cultured for 4 hours;

[0077] PMA group: Add 100 nM PMA to each well and incubate for 4 hours;

[0078] PMA group + Anti-CD53 low, medium and high concentration groups: 100 nM PMA and 10, 20 and 40 ng / mL Anti-CD53 were added to each well and incubated for 4 hours.

[0079] Cells from each group were collected, and the production of MPO and ROS in mouse bone marrow neutrophils was detected by flow cytometry; the results are as follows. Figure 4 As shown in (A), after PMA stimulation, the Anti-CD53 40 ng / mL group showed significantly greater protection against MPO and ROS production compared to other groups. Subsequently, we detected these effects using immunofluorescence and scanning electron microscopy. Figure 4 As shown in (BC), inhibiting CD53 effectively reduced NETs release. Cell supernatants from each group were collected, and the levels of cellular inflammatory cytokines were detected. Figure 4 As shown in (D), the levels of inflammatory factors (IL6, TNF-α, MCP-1) in the Anti-CD53 40 ng / mL group were also significantly lower than those in the PMA group. Figure 5 As shown in (AB), we extracted proteins for Western blotting analysis. The protein level of PAD4 in neutrophils was significantly increased in the PMA group, and decreased after administration of Anti-CD53. The results indicate that inhibiting CD53 significantly reduces NET formation in vitro.

[0080] Example 5: Application of CD53 neutralizing antibody (Anti-CD53) in the prevention / treatment of acute pancreatitis

[0081] I. Acute pancreatitis model induced by taurine in mice

[0082] Experimental animals: 6-8 week old male C57BL / 6J mice (average weight 25g).

[0083] An acute pancreatitis model was established in mice by intraperitoneal injection of baicalin (100 μg / kg, 1 hour apart, 10 injections). Control mice received a strict PBS intraperitoneal injection. Mice underwent in vivo cardiac perfusion to remove circulating blood. Pancreatic tissue was rapidly extracted; some proteins were extracted for Western blotting, while the remaining tissue was fixed, dehydrated, and prepared for paraffin sectioning and eosin staining.

[0084] II. Intraperitoneal injection of Anti-CD53

[0085] Mice were randomly divided into four groups: NC group, NC+Anti-CD53 (160 μg / kg) treatment group, AP group (Cae), and AP+Anti-CD53 treatment groups with different dose gradients (n=7 per group). The Anti-CD53 treatment groups with different dose gradients received intraperitoneal injections of Anti-CD53 (40 μg / kg, 80 μg / kg, 160 μg / kg) after the first injection of lecithin. The Control group received intraperitoneal injections of the same dose of phosphate-buffered saline (PBS) as the other groups. The AP group and the Control+Anti-CD53 (160 μg / kg) treatment group received the same amount of IgG intraperitoneally.

[0086] All mice were sacrificed 12 hours after intraperitoneal injection of the first dose of hygroscopic agent, and serum was collected for serum enzyme level detection. Subsequently, the mice underwent in vivo cardiac perfusion to remove circulating blood, and pancreatic tissue was rapidly extracted, fixed, dehydrated, and prepared for HE-stained paraffin sections and eosin staining.

[0087] The results are as follows Figure 6 As shown in (A) and (D), Anti-CD53 can significantly improve pancreatic tissue damage (pathological damage). Figure 6 As shown in (A) and (C), immunofluorescence detection revealed that the formation of NETs (Cith3 / MPO co-localization) in the pancreatic tissue of mice treated with Anti-CD53 was significantly reduced compared with the AP group. Figure 6 (B) This study showed that Anti-CD53 can reduce serum amylase and lipase levels in acute pancreatitis (AP). Figure 6 As shown in (E), Anti-CD53 also significantly reduced the serum levels of inflammatory factors (IL-6, TNF-α, MCP-1) in AP mice. The results indicate that Anti-CD53 can protect against pancreatic damage in AP mice.

[0088] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of CD53 as a diagnostic marker in the preparation of products for the diagnosis of acute pancreatitis, characterized in that, The products include test strips or test papers, detection chips or reagent kits.

2. The application according to claim 1, characterized in that, The diagnosis of acute pancreatitis includes the following steps: (1) Collect samples from the subjects to be tested and collect control samples; (2) Detect and compare the expression levels of CD53 in the test subject samples and control samples; If the expression level of CD53 in the sample of the test subject is higher than that in the control sample, the test subject is diagnosed with acute pancreatitis or at risk of having acute pancreatitis.

3. The application of CD53 as a prognostic biomarker in the preparation of products for predicting the prognosis of acute pancreatitis, characterized in that, The products include test strips or test papers, detection chips or reagent kits.

4. The application according to claim 3, characterized in that, The prognosis assessment for acute pancreatitis includes the following steps: (1) Samples from patients with post-prognosis acute pancreatitis were collected as the test group, and samples from patients with pre-prognosis acute pancreatitis were collected as the control group; (2) Detect and compare the expression levels of CD53 in the samples of the test group and the control group; If the expression level of CD53 in the test group is lower than that in the control group, the prognosis of the test group is considered to be good.

5. The application according to claim 4, characterized in that, The applicable test samples for patients with acute pancreatitis who are considered to have prognoses and prognoses are blood, plasma, or serum derived from peripheral blood.

6. The application according to claim 5, characterized in that, The appropriate test samples for patients with acute pancreatitis who are considered to have prognostic and pre-prognostic outcomes are neutrophils or serum derived from peripheral blood.

7. The use of CD53 neutralizing antibodies in the preparation of drugs for the prevention and / or treatment of acute pancreatitis, characterized in that, The CD53 neutralizing antibody Anti-CD53 downregulates the expression level of CD53.

8. The application according to claim 7, characterized in that, The drug in question is one that reduces the expression of serum inflammatory factors to alleviate the inflammatory response.

9. The application according to claim 7, characterized in that, The drug is a drug that improves pancreatic tissue damage by inhibiting the formation of extracellular traps of neutrophils.

10. The application according to claim 7, characterized in that, The drug is one that lowers serum amylase and serum lipase levels.

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