CN118370816A - Application of IL-2/anti-IL-2 antibody immune complex - Google Patents

Abstract

The present invention discloses an application of an IL-2/anti-IL-2 antibody immune complex in the preparation of an anti-COPD drug, which can slow down the occurrence and development of COPD. Action site: Anti-IL-2 antibodies block IL-2Rβ and IL-2Rγ receptors on immune cells, and exogenous IL-2 binds to IL-2Rα receptors on Treg cells to selectively amplify Treg cells, thereby regulating immune homeostasis.

Description

Application of IL-2/anti-IL-2 antibody immune complex

Technical Field

The present invention relates to the technical field of biomedicine, and in particular to the use of IL-2/anti-IL-2 antibody immune complexes in the preparation of anti-COPD drugs.

Background technique

Chronic obstructive pulmonary disease (COPD) is a major disease that seriously endangers people's health. The goal of COPD treatment is to alleviate symptoms, prevent disease progression, alleviate or prevent further decline in lung function, and improve mobility. Its treatment is mainly based on smoking cessation and drug therapy. Commonly used drugs include bronchodilators, inhaled glucocorticoids, theophylline and other drugs. However, even under standardized treatment, many patients still experience acute exacerbations of COPD. One of the reasons is that the existing treatment methods are mainly symptom treatments, not targeted drug treatments for chronic inflammation and injury mechanisms. So far, there is a lack of clinical drug treatments that can effectively and significantly slow down or reverse disease progression, which is the main challenge facing the current clinical treatment of COPD. Therefore, there is an urgent clinical need to deeply explore the intrinsic mechanism of the occurrence and development of the disease caused by the interaction between harmful exposure factors and host factors, to find key signaling pathways and druggable targets that are significantly changed during the occurrence and development of the disease, and to develop drugs with clinical transformation prospects.

In recent years, with the further understanding of the pathogenesis and molecular mechanism of COPD and the emphasis on the heterogeneity of COPD, the research and development of COPD drugs has made great progress, especially the research and development of targeted drugs. The drugs currently under development mainly include: (1) Antioxidants. Thiol antioxidants can block mucin disulfide bonds, decompose mucin, dilute sputum, enhance the physiological transport function of the mucociliary system, remove reactive oxygen species, and reduce lipid peroxidation. However, due to their low oral bioavailability and acidic nature, they are not suitable for inhalation, and their clinical application is still controversial. In addition, targeted antioxidants such as SIRT1 activators resveratrol and SRT720 are also being evaluated. (2) Protease inhibitors, such as the recombinant human endogenous trypsin (AT) inhibitor DX-890 and elastin-degrading matrix metalloproteinases (MMPs) and neutrophil elastase (NE) inhibitors AZD1236, ONO-5046, and AZD9668 are under development, but most synthetic MMP inhibitors have been discontinued due to their high toxicity. (3) Chemokine and cytokine inhibitors, including specific pro-inflammatory and pro-neutrophil cytokines and chemokines. (4) PDE4 inhibitors. Selective PDE4 inhibitors such as roflumilast and cilomilast have been developed in clinical practice. In addition, drugs such as IC-485, ADW-2-281, and CHF6001 are also under development. PDE4 inhibitors are often The common gastrointestinal reactions and potential risks to mental health limit its clinical application; (5) Peroxisome proliferator-activated receptor (PPAR) agonists. Activation of PPARγ and PPARα may have anti-inflammatory and immunomodulatory effects. PPARγ can also inhibit pulmonary fibrosis. Current studies have shown that PPAR targeted agonist drugs such as fenofibrate may have therapeutic potential; (6) Phosphoinositide-3 kinase (PI-3Ks) inhibitors. The isomer of PI-3K is PI-3Kγ, which is involved in neutrophil activation and recycling, such as GSK2269557, AS605 Research on drugs such as 240 is in progress; (7) P38 mitogen-activated protein kinase (P38MARK) inhibitors. P38MARK plays an important role in inflammation and tissue construction and is considered to be a therapeutic target. Currently, a variety of P38MARK inhibitors such as SB-203580 and SB-239063 have been developed and have been shown to have good anti-inflammatory effects in COPD animal models; (8) Other targeted regulators, such as adhesion molecule inhibitors, endothelin inhibitors, adenosine A2a receptor agonists and other small molecule regulators, are also being confirmed for their therapeutic effects on COPD.

Summary of the invention

In order to overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide an application of an IL-2/anti-IL-2 antibody immune complex in the preparation of an anti-COPD drug, which can slow down the occurrence and development of COPD.

The technical solution of the present invention is: an application of an IL-2/anti-IL-2 antibody immune complex in the preparation of an anti-COPD drug, wherein the action site is: the anti-IL-2 antibody blocks the IL-2Rβ and IL-2Rγ receptors on immune cells, and exogenous IL-2 binds to the IL-2Rα receptor on Treg cells to selectively amplify Treg cells, thereby playing a role in regulating immune homeostasis.

The present invention provides a new drug for treating COPD, namely, IL-2/anti-IL-2 antibody immune complex, which can slow down the occurrence and development of COPD.

Preferably, the IL-2/anti-IL-2 antibody immune complex effectively expands the number of Treg cells in the lung and spleen.

Preferably, the IL-2/anti-IL-2 antibody immune complex reduces airway and lung inflammatory cell infiltration.

Preferably, the IL-2/anti-IL-2 antibody immune complex reduces inflammatory destruction of lung tissue and mucus secretion.

Preferably, the drug for preventing and treating COPD contains an effective dose of IL-2/anti-IL-2 antibody immune complex and a pharmaceutical excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that IL-2/anti-IL-2 antibody immune complexes can effectively expand the number of Treg cells in the spleen. Figure 1A is a flow cytometry analysis of the proportion of Treg cells in the spleen of different groups of mice, and Figure 1B is a statistical graph of the proportion of Treg cells in the spleen of each group of mice.

Figure 2 shows that IL-2/anti-IL-2 antibody immune complexes can effectively amplify the number of Treg cells in the lungs. Figure 2A is a statistical diagram of the proportion of Treg cells in the lungs of different groups of mice detected by immunofluorescence, with arrows indicating Treg cells, and Figure 2B is a statistical diagram of the proportion of Treg cells in the lungs of each group of mice.

Figure 3 shows that IL-2/anti-IL-2 antibody immune complexes can reduce airway inflammatory cell infiltration. Figure 3A shows a smear of bronchoalveolar lavage fluid and Swiss staining to identify different types of inflammatory cells, Figure 3B shows the total cell count in bronchoalveolar lavage fluid, Figure 3C shows the number of neutrophils in bronchoalveolar lavage fluid smears, and Figure 3D shows the number of macrophages in bronchoalveolar lavage fluid smears.

Figure 4 shows that IL-2/anti-IL-2 antibody immune complexes can reduce lung inflammatory cell infiltration. Figure 4A uses immunofluorescence to mark neutrophils and macrophages in mouse lung tissue sections and counts them, Figure 4B is a statistical chart of neutrophil counts, and Figure 4C is a statistical chart of macrophage counts.

Figure 5 shows that IL-2/anti-IL-2 antibody immune complex can reduce airway inflammation damage. Figure 5A is a lung tissue stained with HE method and shows the damage of airway inflammation around the lung tissue, and Figure 5B is a scoring method to evaluate the degree of damage of airway inflammation around the lung tissue of mice in different groups.

Figure 6 shows that IL-2/anti-IL-2 antibody immune complex can reduce inflammatory damage in lung tissue. Figure 6A shows the lung tissue stained by HE method and shows the inflammatory damage around the alveoli, and Figure 6B shows the degree of inflammatory damage around the alveoli in the lung tissue of mice in different groups evaluated by scoring method.

Figure 7 shows that IL-2/anti-IL-2 antibody immune complexes can reduce airway mucus secretion. Figure 7A is the staining of the airway of lung tissue of different groups of mice using AB-PAS staining method, and Figure 7B is the evaluation of airway mucus secretion of lung tissue of different groups of mice using scoring method.

Detailed ways

The present invention provides a new drug for treating COPD, namely, IL-2/anti-IL-2 antibody immune complex, which can slow down the occurrence and development of COPD. An application of IL-2/anti-IL-2 antibody immune complex in the preparation of anti-COPD drugs, with action sites: anti-IL-2 antibodies block IL-2Rβ and IL-2Rγ receptors on immune cells, and exogenous IL-2 binds to IL-2Rα receptors on Treg cells to selectively amplify Treg cells, thereby regulating immune homeostasis.

The embodiments of the present invention are described in detail below.

Male C57BL/6 mice (8 weeks old, 20–25 g) were purchased from Sibeifu, Beijing, China, and housed under a constant temperature of 23 °C, 60% humidity, and a 12 h/12 h light-dark cycle. The animals had free access to water and food. The mice were randomly divided into groups, with 6–8 mice in each group. The COPD model was induced by intratracheal infusion of lipopolysaccharide followed by exposure to cigarette smoke. Briefly, mice were anesthetized by inhalation of isoflurane and intratracheally infused with LPS (1 g/L, 20 L) on days 0 and 14. After each LPS treatment, the mice were placed in a sealed box and exposed to smoke from 10 unfiltered commercial cigarettes three times a day for a total of 28 days of smoke exposure. Mice in the control group received similar treatment but were exposed to air. All experimental protocols were approved by the Animal Research Ethics Committee of the China-Japan Friendship Hospital (No. Zryhyy12-20-01-12).

According to the optimal dose/ratio and dosing interval of IL-2C reported in previous studies, 1 μg IL-2 was mixed with 5 μg anti-IL-2 monoclonal antibody at room temperature for 30 min to prepare IL-2C, and 5 μg IgG was used per mouse to prepare isotype control. Mice were intraperitoneally injected with IL-2C or isotype control every 2 weeks for 3 consecutive days.

(1) IL-2/anti-IL-2 antibody immune complexes can effectively expand the number of Treg cells in the lungs and spleen

To clarify the role of IL-2/anti-IL-2 antibody immune complexes, the proportion of Treg cells in different groups of mice was first evaluated. Fresh spleen tissues from different groups of mice were collected, ground, digested, and made into single cell suspensions. After centrifugation at 300g for 5 minutes, they were resuspended in 1ml PBS. Each tube was incubated with 2μL FC blocking antibody for 10 minutes, and live/dead dye was added to detect cell activity. After that, anti-CD3, anti-CD4, anti-CD25 and anti-FOXP3 antibody combinations were added to detect Tregs, incubated in the dark for 30 minutes, centrifuged, and then suspended in 300μL PBS and detected using a FACS Calibur flow cytometer (Beckman Cytoflex, Germany). The results showed that IL-2/anti-IL-2 antibody immune complexes significantly increased the percentage of CD4+CD25+Foxp3+T cells in the CD4+T cell population in the spleen (Figure 1).

The proportion of Treg cells in lung tissue was detected by immunofluorescence. The specific implementation scheme was as follows: the left lung of each mouse was paraffin-embedded and 3 μm thick sections were made. Endogenous peroxidase was blocked with Tris/EDTA buffer (pH 9.0), 3% hydrogen peroxide, and nonspecific antibodies were inhibited with 10% goat serum. Antibodies against FOXP3, Ly6g, and F4/80 were then added, and the cells were incubated with antibodies overnight. Subsequently, the cells were stained with fluorescent secondary antibodies for 1 hour at room temperature. The tissue was sealed with an anti-fluorescence quencher containing DAPI and observed under a confocal microscope (NIKON AIR, Japan). The results also confirmed that the proportion of CD4+CD25+Foxp3+Treg cells in the lungs of IL-2C mice was significantly increased (Figure 2). The results of the study indicate that Treg cells amplified in the spleen of IL-2/anti-IL-2 antibody immune complexes may be recruited to the lungs to play an anti-inflammatory role.

(2) IL-2/anti-IL-2 antibody immune complexes can reduce airway and lung inflammatory cell infiltration

To evaluate the improvement of inflammation in mice by IL-2/anti-IL-2 antibody immune complexes, the total number and classification of cells in BALF were counted. The specific implementation plan is: after the COPD modeling scheme is completed, the mice are anesthetized by intraperitoneal injection of amobarbital (200 mg/kg). 0.8 mL of sterile saline is given through tracheal intubation and slowly aspirated 5 times. About 0.6 mL of BALF was recovered and centrifuged at 1000 rpm for 5 minutes at 4°C. The cell pellet was resuspended in 1 ml of phosphate-buffered saline (PBS) and the cells were counted using an automatic cell counter. 100 L of smear was taken from the cell suspension, 200 cells were classified by Wright-Giemsa staining, and observed under an optical microscope (OLYMPUSBX 53, Japan).

The BALF inflammatory cells of mice in different treatment groups were stained to detect the presence of lung inflammatory cells, and it was found that the IL-2/anti-IL-2 antibody immune complex could significantly reduce the number of airway neutrophils and macrophages (Figure 3). The number of neutrophils and macrophages in the alveoli was analyzed by immunofluorescence staining of lung tissue, and the results showed that the IL-2/anti-IL-2 antibody immune complex could effectively reduce the infiltration of inflammatory cells in lung tissue (Figure 4).

(3) IL-2/anti-IL-2 antibody immune complex can reduce lung tissue inflammation and mucus secretion

The effects of IL-2/anti-IL-2 antibody immune complexes on lung tissue morphology and pathology were evaluated by pathological staining of mouse lung tissue, including hematoxylin and eosin (H&E) and alcian blue-periodic acid-Schiff (AB-PAS) staining, as follows: The left lung of mice was fixed in 4% formalin buffer, and paraffin sections of lung tissue were prepared, followed by H&E and AB-PAS staining. Morphometric quantification of stained lung sections was performed using a custom-made digital image processing system. Bronchi and parenchyma were examined for blinded histopathology as described in previous studies. The severity of inflammation was scored on a scale of 0–3, defined as follows: 0 = no inflammatory cells; 1 = mild inflammation with inflammatory cell foci in the bronchial or vascular walls and alveolar septa; 2 = moderate, patchy, or localized inflammation in the bronchial or vascular walls and alveolar septa, affecting less than one-third of the total cross-sectional area of the lung; and 3 = severe inflammation with diffuse inflammatory cells in the bronchial or vascular walls and alveolar septa, affecting one-third to two-thirds of the lung area. On H&E-stained lung tissue images, the severity of peribronchial inflammation was quantified from 0 to 5 based on the number of inflammatory cells, where 0 = no cells, 1 = a small number of cells, 2 = a cell layer with a depth of one cell layer, 3 = a cell ring with a depth of two cells, 4 = a cell ring with a depth of three to four cells, and 5 = a cell ring with a depth of five or more cells. The histological mucus index of AB-PAS-stained specimens was determined, with positive airway goblet cells (purple). Specifically, the number of airway goblet cells in each field of view was quantitatively determined, with a score of 0-4 reflecting the degree of AB-PAS positive staining, 0 = no goblet cells; 1 = goblet cells account for less than 25% of the total number of airway epithelial cells; 2 = goblet cells account for 26-50% of the airway epithelial cells; 3 = goblet cells account for 51-75% of the airway epithelial cells; 4 = goblet cells account for more than 75% of the airway epithelial cells.

HE staining showed that IL-2/anti-IL-2 antibody immune complexes could significantly improve inflammatory damage to lung tissue (Figures 5-6), and AB-PAS staining showed that IL-2/anti-IL-2 antibody immune complexes could inhibit airway goblet cell proliferation and airway mucus secretion (Figure 7). These results support that IL-2/anti-IL-2 antibody immune complexes can reduce cigarette smoke-induced lung inflammatory damage.

The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the protection scope of the technical solution of the present invention.

Claims (5)

1. The application of an IL-2/anti-IL-2 antibody immune complex in preparing anti-chronic obstructive pulmonary drug is characterized in that: site of action: the anti-IL-2 antibody seals IL-2Rbeta and IL-2Rgamma receptors on immune cells, and exogenous IL-2 binds with IL-2Ralpha receptors on Treg cells to selectively expand the Treg cells, thereby playing a role in regulating immune homeostasis.
2. 2. The use of an IL-2/anti-IL-2 antibody immune complex according to claim 1 for the manufacture of an anti-chronic obstructive pulmonary drug, wherein: the IL-2/anti-IL-2 antibody immune complex is effective in expanding the number of Treg cells in the lung and spleen.
3. 3. The use of an IL-2/anti-IL-2 antibody immune complex according to claim 1 for the manufacture of an anti-chronic obstructive pulmonary drug, wherein: the IL-2/anti-IL-2 antibody immune complex reduces inflammatory cell infiltration in the airways and lungs.
4. 4. The use of an IL-2/anti-IL-2 antibody immune complex according to claim 1 for the manufacture of an anti-chronic obstructive pulmonary drug, wherein: the IL-2/anti-IL-2 antibody immune complex reduces inflammatory destruction and mucus secretion in lung tissue.
5. 5. Use of an IL-2/anti-IL-2 antibody immune complex according to any one of claims 1-4 for the preparation of an anti-chronic obstructive pulmonary drug, characterized in that: the medicine for preventing and treating chronic obstructive pulmonary disease contains effective dose of IL-2/anti-IL-2 antibody immune complex and medicinal excipient.