Antibody for evaluating curative effect of pediatric pneumonia asthma and application thereof

Abstract

The application belongs to the technical field of biomedicine, and discloses an antibody for evaluating the curative effect of pediatric pneumonia asthma and application thereof. The heavy chain CDR region sequence of the monoclonal antibody (hybridoma cell strain 2B5 secretion) is SEQ ID NO: 2-4, and the light chain CDR region sequence is SEQ ID NO: 6-8. The dissociation constant of the antibody with IL-5R antigen reaches 2.435*10 ‑ ¹ 0 mol / L, and the affinity is high. The antibody can accurately evaluate the curative effect of pediatric pneumonia asthma by detecting lung function, bronchoalveolar lavage fluid eosinophil proportion and lung tissue inflammation, and can significantly improve airway obstruction and inflammation of the asthma model mouse, and the effect is better than that of dexamethasone. The problems of lack of specific tools for curative effect evaluation and large side effects of treatment drugs are solved, and a new scheme is provided for curative effect evaluation and adjuvant treatment of pediatric pneumonia asthma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a monoclonal antibody against human interleukin-5 receptor α subtype (IL-5Rα), its preparation method, and its application in evaluating the efficacy of products for treating pediatric pneumonia and asthma. It can also be used as an adjunct therapy for pediatric pneumonia and asthma, providing specific molecular tools and new strategies for the diagnosis and treatment of pediatric pneumonia and asthma. Background Technology

[0002] Childhood pneumonia and asthma are common chronic respiratory diseases in childhood, with a global incidence rate of 5%-10% in children under 5 years old, and this rate has been increasing year by year in recent years. The disease is characterized by chronic airway inflammation, hypermucus secretion, and airway hyperresponsiveness. Clinical manifestations include recurrent cough, wheezing, and chest tightness. Acute exacerbations can lead to respiratory failure. Long-term recurrent attacks not only affect children's lung function development but also cause growth retardation and psychological problems, placing a heavy medical burden on families and society. Currently, clinical diagnosis mainly relies on symptoms and lung function tests. However, early inflammatory markers are atypical, easily confused with ordinary pneumonia, delaying intervention.

[0003] Current treatments for pediatric pneumonia and asthma primarily focus on controlling inflammation, with commonly used medications including corticosteroids (such as dexamethasone) and bronchodilators. While corticosteroids can provide short-term relief from inflammation, long-term use can suppress the development of the child's immune system, leading to side effects such as growth inhibition and osteoporosis, especially posing a higher safety risk to infants under 3 years old. Furthermore, some patients exhibit hormone resistance, resulting in poor treatment outcomes, and there is a lack of specific treatments targeting the core inflammatory pathways of the disease (such as the IL-5 / IL-5R-mediated eosinophil recruitment pathway), making it difficult to fundamentally control the progression of inflammation.

[0004] In terms of efficacy evaluation, current clinical practice mainly relies on symptom relief, pulmonary function indicators (such as airway resistance and dynamic compliance), and routine inflammatory cell counts (such as peripheral blood eosinophils) to determine efficacy. However, these indicators have limitations: symptom evaluation is easily influenced by subjective factors, routine inflammatory cell counts cannot accurately reflect the local inflammatory state of the airways, and there is a lack of specific evaluation tools targeting the IL-5 / IL-5R pathway. This makes it difficult to determine early and accurately whether treatment has blocked the core pathological links of the disease, resulting in some patients experiencing symptom relief but persistent airway inflammation, increasing the risk of disease recurrence. Therefore, developing a molecular tool that specifically targets the IL-5R pathway and has both efficacy evaluation and adjuvant therapy functions is key to solving the current dilemma in the diagnosis and treatment of pediatric pneumonia and asthma. Summary of the Invention

[0005] To address the aforementioned issues, this invention first provides a monoclonal antibody for evaluating the efficacy of treatments for pediatric pneumonia and asthma. The heavy chain complementarity-determining region (CDR1) of this monoclonal antibody has the sequences SEQ ID NO:2, CDR2, and CDR3 as SEQ ID NO:4, and the light chain complementarity-determining region (CDR1) has the sequences SEQ ID NO:6, CDR2 as SEQ ID NO:7, and CDR3 as SEQ ID NO:8. Furthermore, the heavy chain variable region (VH) of this monoclonal antibody has the sequence SEQ ID NO:1, and the light chain variable region (VL) has the sequence SEQ ID NO:5. It is secreted by hybridoma cell line 2B5 and purified to ≥98% purity using a Protein G affinity chromatography column.

[0006] In some embodiments, the monoclonal antibody binds to the IL-5R antigen with high affinity, and its dissociation constant KD is 2.435 × 10⁻⁶. -10 It has a concentration of mol / L and specifically binds to human IL-5Rα, without cross-reacting with other cytokine receptors. Furthermore, the specificity of this monoclonal antibody can be verified by Western blot (anti-His tag antibody), and it shows a single clear band when detected by SDS-PAGE.

[0007] The present invention also provides a method for preparing the above-mentioned monoclonal antibody, the method comprising the following steps: Preparation of human IL-5R active antigen fragment: Based on the human IL-5Rα subtype 4 precursor sequence recorded in NCBI Reference Sequence: NP_001230028.1, primers with restriction enzyme sites were designed using the extracellular active region (SEQ ID NO:1). PCR amplification was performed using cDNA containing this sequence as a template. The amplified product was ligated after double digestion with the pET-28a (+) vector, transformed into *E. coli* DH5α, and recombinant expression vectors were screened. The resulting fragments were then transformed into *E. coli* BL21 (DE3) to induce expression, purified by Ni-NTA affinity chromatography, dialyzed, and lyophilized to obtain the human IL-5R active antigen fragment. Screening of immunized mice and cell fusion: Human IL-5R active antigen fragments were emulsified with adjuvant and immunized with 6-8 week old female BALB / c mice. Antiserum titers were measured after multiple immunizations, and mice with a titer ≥1:10 were selected. 5 Mice were subjected to shock immunization; mouse spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 5:1, fused with PEG 1500, and screened on HAT medium. Positive wells were selected by indirect ELISA after 14 days (OD). 450With a ratio > 3, the hybridoma cell line 2B5, which stably secretes antibodies, was obtained by cloning and culturing three times using the limiting dilution method. Antibody purification and identification: Pretreated BALB / c mice were intraperitoneally inoculated with hybridoma cell line 2B5. Ascites fluid was collected, filtered through a 0.22 μm filter, and loaded onto a Protein G affinity chromatography column. The column was equilibrated with phosphate buffer, eluted with glycine-HCl, and neutralized with Tris-HCl to obtain purified antibodies. Purity was determined by SDS-PAG (≥98%), concentration by BCA method (5-8 mg / mL), and titer by indirect ELISA (≥1:10). 6 The total RNA from hybridoma cells was extracted and reverse transcribed into cDNA, and the VH and VL genes were amplified and sequenced to confirm the CDR region sequence.

[0008] Finally, this invention provides the application of the above-mentioned monoclonal antibody, which is its use in the preparation of products for evaluating the efficacy of pediatric pneumonia and asthma treatment, and can also be used as an adjunct treatment for pediatric pneumonia and asthma.

[0009] In some embodiments, the product for evaluating the efficacy of treatment for pediatric pneumonia and asthma is evaluated by detecting at least one of the following in pediatric pneumonia and asthma patients or disease models: lung function (including airway resistance Ri and dynamic compliance Cdyn), the proportion of eosinophils in bronchoalveolar lavage fluid, and the degree of lung tissue inflammation (determined by an inflammation score of 0-5 after HE staining). The adjuvant therapy targets IL-5R with the monoclonal antibody, blocking the eosinophil-mediated inflammatory response. In an asthmatic mouse model, it can reduce airway resistance by approximately 46%, increase dynamic compliance by approximately 82%, and reduce the proportion of eosinophils by approximately 86%, showing better therapeutic effects than dexamethasone.

[0010] Compared with the prior art, the present invention has at least the following beneficial effects: High specificity: The monoclonal antibody of this invention precisely targets human IL-5Rα, with a dissociation constant of 10. -10 At the mol / L level, it can specifically capture the inflammatory state of the IL-5 / IL-5R pathway, solving the problem that existing efficacy evaluation indicators (such as subjective symptoms and conventional inflammatory cell counts) cannot accurately reflect the core pathological links in the airway. Balancing efficacy and safety: When used as an adjunct therapy for childhood pneumonia and asthma, this antibody can significantly improve lung function and suppress inflammation, without the growth inhibition and immunosuppression side effects caused by glucocorticoids (such as dexamethasone), thus avoiding the safety risks of existing treatment drugs; Multifunctional: The monoclonal antibody of this invention has the dual functions of efficacy evaluation and adjuvant therapy. It provides a precise molecular tool for efficacy evaluation in clinical practice and can also be used as a targeted therapy, breaking through the dual dilemma of "inaccurate evaluation and limited treatment" in the current diagnosis and treatment.

[0011] This invention is the first to realize the development of an integrated "evaluation-treatment" molecular tool targeting the IL-5 / IL-5R core pathway in pediatric pneumonia and asthma, providing a new specific strategy for the precision diagnosis and treatment of pediatric pneumonia and asthma, and filling the gap in the field of targeted diagnostic and therapeutic tools. Attached Figure Description

[0012] Figure 1 The images show the SDS-PAGE and Western blot results of the human IL-5R active antigen fragment. The left side shows the SDS-PAGE electrophoresis image, which shows that the target protein has a molecular weight of approximately 38 kDa and a purity of > 95%. The right side shows the Western blot results, which were detected with anti-His tag antibody. The target protein band is clear, proving that the antigen fragment carries a His tag and has the correct specificity.

[0013] Figure 2 Absorbance (OD) of positive hybridoma wells screened by indirect ELISA 450 Results figure; the horizontal axis represents the hybridoma cell line number, and the vertical axis represents OD. 450 Value; the results showed that the OD value of strain 2B5 was... 450 The value was significantly higher than other lines, and the ratio with the negative control (SP2 / 0 cell supernatant) was > 3, indicating that it was a positive hybridoma cell line.

[0014] Figure 3 The sensor graph of Biacore T200 for detecting the binding of monoclonal antibody 2B5 to IL-5R antigen; the horizontal axis is time (seconds) and the vertical axis is the response value (RU); the curve shows that the response value rises rapidly after the antibody binds to the antigen, and the response value decreases slowly during the dissociation phase, which conforms to the 1:1 Langmuir binding model and can be used to calculate the binding constant (Ka), dissociation constant (Kd) and affinity constant (KD). Detailed Implementation

[0015] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0016] Example 1: Preparation of human IL-5R active antigen fragment Based on the human IL-5Rα isoform 4 precursor sequence described in the NCBI Reference Sequence: NP_001230028.1 (interleukin-5 receptor subunit alpha isoform 4 precursor [Homosapiens]), its extracellular active region was selected as the target antigen fragment based on functional domain prediction. Upstream and downstream primers were designed. The upstream primer introduced an NcoI restriction site, and the downstream primer introduced an XhoI restriction site and a 6×His tag sequence. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0017] The target antigen fragment sequence is as follows: DEKISLLPPVNFTIKVTGLAQVLLQWKPNPDQEQRNVNLEYQVKINAPKEDDYETRITESKCVTILHKGFSASVRTILQNDHSLLASSWASAELHAPPGSPGTSIVNLTCTTNTTEDNYSRLRSYQVSLHCTWLVGTDAPEDTQYFLYYRYGSWTEECQEYSKDTLGR NIACWFPRTFILSKGRDWLAVLVNGSSKHSAIRPFDQLFALHAIDQINPPLNVTAEIEGTRLSIQWEKPVSAFPIHCFDYEVKIHNTRNGYLQIEKLMTNAFISIIDDLSKYDVQVRAAVSSMCREAGLWSEWSQPIYVGNDEHKPLREWFVIVIMATHHHHHH (SEQ ID NO:9).

[0018] Using cDNA containing the full-length sequence of SEQ ID NO:1 as a template, PCR amplification was performed using PrimeSTAR HS DNA polymerase. The reaction mixture (50 μL) consisted of: 1 μL template cDNA, 1 μL each of forward and reverse primers, 25 μL 2×PCR Mix, and 2 μL ddH2O. Reaction conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 58℃ annealing for 15 s, and 72℃ extension for 45 s, for a total of 35 cycles; and a final extension at 72℃ for 5 min. The amplified products were verified by 1% agarose gel electrophoresis, and the target fragment was recovered.

[0019] The recovered target fragment and the pET-28a (+) vector (Novagen) were double-digested with NcoI and XhoI (NEB) at 37℃ for 4 h. After gel recovery, the digestion products were ligated overnight with T4 DNA ligase (Promega) at 16℃. The ligation product was transformed into E. coli DH5α competent cells (Tiangen Biotech), plated on LB agar plates containing 50 μg / mL kanamycin, and cultured at 37℃ for 16 h. Single colonies were picked and cultured, and plasmids were extracted, verified by double enzyme digestion and sequencing (BGI Genomics), yielding the recombinant expression vector pET-28a-IL5R-His.

[0020] The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells, and positive clones were screened and inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking until OD. 600 When the concentration of the IL-5R molecule is 0.6-0.8, add IPTG to a final concentration of 0.5 mM and induce at 30°C for 6 h. Centrifuge the induced bacterial culture (8000×g, 10 min), collect the bacterial pellet, and store at -80°C. Resuspend the bacterial pellet in PBS (pH 7.4), sonicate (300W, 3 s operation, 5 s interval, 30 min total), centrifuge at 4°C (12000×g, 20 min), and collect the supernatant. Filter the supernatant through a 0.22 μm filter membrane and load it onto a Ni-NTA affinity chromatography column (GE Healthcare). Equilibrate with PBS containing 20 mM imidazole, and elute the target protein with PBS containing 500 mM imidazole. The purity of the eluent is determined by SDS-PAGE (>95%), and specificity is verified by Western blot (anti-His tag antibody). After dialysis to remove imidazole, freeze-dry to obtain the human IL-5R active antigen fragment. Figure 1 .

[0021] Figure 1 The results showed that the molecular weight of the human IL-5R active antigen fragment was approximately 38 kDa and it carried an HIS tag, which was in line with expectations.

[0022] Example 2: Preparation of monoclonal antibodies against human IL-5R active antigen fragments The human IL-5R active antigen fragment prepared in Example 1 was diluted to 100 μg / 100 μL with PBS, emulsified with an equal volume of Freund's complete adjuvant (Sigma), and intraperitoneally injected into 6-8 week old female BALB / c mice, 100 μL per mouse. A second immunization was performed 2 weeks later, using the same antigen, emulsified with an equal volume of Freund's incomplete adjuvant, at the same dose. Booster immunizations were then performed every 2 weeks for a total of 3 times. On day 7 after the last immunization, blood was collected from the orbital sinus, serum was separated, and antiserum titer was detected using indirect ELISA: 96-well plates were coated with 5 μg / mL IL-5R antigen, serially diluted serum was incubated, and HRP-labeled goat anti-mouse IgG (1:5000, Jackson) was used for color development, OD... 450 A titer >0.5 and more than 3 times that of the negative control (normal mouse serum) was considered positive. A titer ≥1:10 was selected. 5 Mice were used for fusion.

[0023] Three days prior to fusion, selected mice were intraperitoneally immunized with an adjuvant-free IL-5R antigen (50 μg / mouse). SP2 / 0 myeloma cells in logarithmic growth phase were washed twice with RPMI 1640 medium. Simultaneously, immunized mice were sacrificed, and spleens were aseptically harvested. Spleen cell suspensions were prepared by grinding and filtration, and trypan blue staining was used to count viable cells > 95%. Spleen cells were mixed at a ratio of 5:1 to myeloma cells, centrifuged at 1000×g for 5 min, and the supernatant was discarded. 1 mL of 50% PEG 1500 (Roche) was slowly added, and the mixture was incubated at 37°C for 1 min. The reaction was terminated by adding RPMI 1640 medium. The pellet was resuspended in HAT selection medium containing 20% ​​fetal bovine serum and seeded into 96-well plates, incubated at 37°C with 5% CO2. After 10 days, the medium was changed to HT medium. On day 14, positive wells were screened using indirect ELISA: the cells were coated with IL-5R antigen (ELITE-MEDIA, recombinant human IL-5 receptor subunit α, catalog number: Q01344). The supernatant from the detection wells was compared with the negative control (SP2 / 0 cell supernatant). 450 A ratio > 3 indicates a positive result. See Figure 2 .

[0024] Figure 2 The results showed that the hybridoma cell line with the highest stable antibody secretion was obtained by cloning the positive wells using the limiting dilution method and cloning them three times in a row. This line was named 2B5.

[0025] 8-10 week old BALB / c mice were pretreated with 0.5 mL of norphyrane (Sigma) via intraperitoneal injection for 7 days. Each mouse was also inoculated with 1 × 10⁻⁶ oz. 6Two B5 positive hybridoma cells were collected, and ascites fluid was collected after 7-10 days. The ascites fluid was centrifuged at 3000×g for 15 min to remove the precipitate, and the supernatant was stored at -80℃. The ascites fluid was then purified using a Protein G affinity chromatography column (GE Healthcare): the ascites fluid was filtered through a 0.22 μm filter and loaded onto the column. The column was equilibrated with 20 mM phosphate buffer (pH 7.0), eluted with 0.1 M glycine-HCl (pH 2.7), and the elution buffer was neutralized with 1 M Tris-HCl (pH 9.0). SDS-PAGE analysis showed that the purified antibody purity was >98%, the concentration determined by BCA method was 5-8 mg / mL, and the indirect ELISA titer was ≥1:10. 6 .

[0026] Total RNA was extracted from hybridoma cell line 2B5 using TRIzol reagent (Invitrogen), and cDNA was synthesized by reverse transcription (PrimeScript RT kit, TaKaRa). Primers were designed based on the mouse IgG constant region sequence, and the heavy chain variable region (VH) and light chain variable region (VL) genes were amplified by PCR. The amplified products were cloned into the pMD19-T vector (TaKaRa), and the sequences were analyzed after sequencing (BGI Genomics) to determine the CDR regions: heavy chain CDR1 (SEQ ID NO:2), CDR2 (SEQ ID NO:3), CDR3 (SEQ ID NO:4); light chain CDR1 (SEQ ID NO:6), CDR2 (SEQ ID NO:7), CDR3 (SEQ ID NO:8).

[0027] Table 1. Sequence analysis of the heavy and light chain variable regions of monoclonal antibody 2B5

[0028] Example 3: Affinity detection of monoclonal antibody 2B5 The CM5 chip was loaded into a Biacore T200 and equilibrated with HBS-EP+ at a flow rate of 30 μL / min until baseline stability was achieved. EDC and NHS (1:1 volume ratio, final concentration 0.2 M each) were mixed, and 40 μL was injected to activate the carboxyl surface of the chip. Goat anti-mouse IgG Fc fragment antibody was diluted to 20 μg / mL with 10 mM sodium acetate (pH 5.0), and 60 μL was injected to immobilize the chip surface, controlling the immobilization level RU. 40 μL of ethanolamine-HCl was injected to block unreacted sites. At a flow rate of 30 μL / min, 1 μg / mL of 2B5 antibody was injected first (120 s) to capture the 2B5 on the chip surface; then IL-5R antigen was injected. After each cycle, 30 μL of glycine-HCl was injected to regenerate the chip (30 s) to restore baseline. A 1:1 Langmuir binding model was fitted using Biacore Evaluation 3.0 software to calculate the dissociation constant between 2B5 and IL-5R antigen, as shown below. Figure 3 And Table 2.

[0029] Table 2. Results of Affinity Detection of Monoclonal Antibody 2B5

[0030] Example 4: Experimental model of monoclonal antibody 2B5 for evaluating the efficacy of treatment for pediatric pneumonia and asthma. Six- to eight-week-old SPF-grade BALB / c mice (female, weighing 18-22g) were used to construct a classic allergic asthma model, simulating the airway inflammation, mucus hypersecretion, and airway hyperresponsiveness characteristics of childhood pneumonia asthma.

[0031] Sensitization phase (days 0 and 7): Each mouse was injected intraperitoneally with 0.2 mL of sensitization solution. The sensitization solution was prepared by mixing OVA (10 μg) with Al(OH)3 adjuvant (2 mg). The adjuvant enhanced the immunogenicity of OVA and induced an allergic reaction in the body.

[0032] Triggering phase (days 14-20): Mice were placed in a nebulizer and nebulized with a 2.5% OVA saline solution for 30 minutes daily. This was repeated for 7 consecutive days to induce persistent inflammation in the airways, leading to an asthma-like pathological state.

[0033] Model validation (day 21): Three mice in the model group were randomly selected to detect airway hyperresponsiveness and the proportion of eosinophils in bronchoalveolar lavage fluid (BALF) to confirm successful model construction (eosinophil proportion ≥5%, significantly increased airway hyperresponsiveness).

[0034] Thirty mice that successfully modeled the disease were randomly divided into three groups (n=10), and ten normal mice were set up as a normal control group. The specific grouping and treatment are as follows: Normal control group: No sensitization or provocation throughout the entire process, and an equal volume of normal saline was injected intraperitoneally on days 14-20; Model control group: Sensitization and challenge were completed, and an equal volume of normal saline was injected intraperitoneally on days 14-20; Experimental group: Sensitization and challenge were completed, and 2B5 (5mg / kg) was injected intraperitoneally on days 14-20. Positive control group: Sensitization and challenge were completed, and dexamethasone (0.5 mg / kg) was injected intraperitoneally on days 14-20.

[0035] Lung function tests: Airway resistance (Ri) and dynamic compliance (Cdyn) were measured using a non-invasive pulmonary function instrument (EMMS R&C Animal Airway Resistance and Lung Compliance Testing System, UK), as shown in Table 3.

[0036] Table 3. Results of Lung Function Tests ( (n=10)

[0037] Note: ## indicates that compared with the normal control group, P < 0.01; ** indicates that compared with the model control group, P < 0.01.

[0038] The results in Table 3 show that, compared with the model group, 2B5 treatment significantly reduced airway resistance by about 46% and improved dynamic compliance by about 82%, with better therapeutic effects than dexamethasone, proving that 2B5 can effectively relieve airway obstruction and stiffness.

[0039] Inflammatory cell analysis of bronchoalveolar lavage fluid (BALF): BALF was collected after mice were sacrificed, and cell counting and classification were performed, with a focus on detecting the proportion of eosinophils, as shown in Table 4.

[0040] Table 4. BALF inflammatory cell count ( , n=10, ×10 4 / mL)

[0041] Note: ## indicates that compared with the normal control group, P < 0.01; ** indicates that compared with the model control group, P < 0.01.

[0042] The results in Table 4 show that the proportion of EOS in the model group increased sharply, while the 2B5 treatment group reduced it significantly by 86%, which fully demonstrates the core role of the antibody in this invention in targeting the IL-5R pathway and inhibiting EOS recruitment.

[0043] HE staining of lung tissue: SPF-grade BALB / c mice from normal control group, model control group, experimental group (treated with 2B5 antibody) and positive control group (treated with dexamethasone) were sacrificed and their intact lung tissue was quickly separated. The middle lobe of the left lung was selected (to ensure consistency of sample location). After removing the connective tissue, the lung tissue was placed in 4% paraformaldehyde fixative and fixed at 4°C for 12-24 hours. The fixative was changed once during the fixation period to ensure adequate fixation.

[0044] Fixed lung tissue was dehydrated using a gradient of ethanol (70% ethanol for 2 hours → 80% ethanol for 2 hours → 95% ethanol for 2 hours → 100% ethanol twice, 1.5 hours each time), cleared with xylene (twice, 1 hour each time), then immersed in melted paraffin (60℃) three times, 1.5 hours each time. Finally, the tissue was embedded in a paraffin block and cooled to solidify. The paraffin block was cut into 4-5 μm thick sections using a rotary microtome, mounted on poly-L-lysine-coated slides, and baked at 60℃ for 2 hours to prevent slide detachment. The tissue was then dewaxed to water (xylene twice, 10 minutes each → 100% → 95% → 80% → 70% ethanol, 5 minutes each → distilled water, stained with hematoxylin for 5-8 minutes, differentiated with 1% hydrochloric acid ethanol for 30 seconds, blued with 0.5% ammonia for 1 minute, rinsed with distilled water, and stained with eosin for 2-3 minutes. After staining, the sections were dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. Under an optical microscope, the bronchial-vascular region was located under low magnification (100×), and the inflammatory cell infiltration, airway wall thickness, and mucus plugs in the lumen were observed under high magnification (400×). A double-blind method was used to score the inflammation on a scale of 0-5 (0 points for no inflammation and 5 points for severe inflammation). The differences in scores among the groups were statistically analyzed to verify the effect of 2B5 antibody on improving lung tissue inflammation.

[0045] Model control group: Extensive and dense inflammatory cell infiltration was observed around the bronchi and blood vessels, the airway walls were significantly thickened, and mucus plugs were visible in the lumen.

[0046] Experimental group: only mild, scattered inflammatory cell infiltration was observed, airway wall thickness was significantly reduced, and the lumen was basically unobstructed.

[0047] Inflammation scores were assessed using a blinded method (0-5 points). The score in the model group was 4.2±0.4, while the score in the 2B5 antibody treatment group decreased to 1.5±0.3 (P < 0.01), directly confirming the superior anti-inflammatory effect of 2B5 at the histological level.

[0048] The above experimental data fully demonstrate that the monoclonal antibody 2B5 of this invention exhibits significant therapeutic effects in a mouse model of pediatric pneumonia and asthma. By specifically targeting IL-5R, it effectively blocks eosinophil-mediated inflammatory cascades, specifically manifested in: significantly improved lung function, potent inhibition of eosinophil infiltration in the lungs, and significant improvement in pathological damage to lung tissue. Therefore, monoclonal antibody 2B5 has enormous application potential and development value in the evaluation and treatment of pediatric pneumonia and asthma.

[0049] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An anti-IL-5R antibody for evaluating the efficacy of treatments for pediatric pneumonia and asthma, characterized in that, The heavy chain complementarity-determining region (CDR1) sequence of the antibody is SEQ ID NO:2, CDR2 sequence is SEQ ID NO:3, and CDR3 sequence is SEQ ID NO:4; the light chain complementarity-determining region (CDR1) sequence is SEQ ID NO:6, CDR2 sequence is SEQ ID NO:7, and CDR3 sequence is SEQ ID NO:

8.

2. The antibody according to claim 1, characterized in that, The heavy chain variable region VH sequence of the antibody is SEQ ID NO:1, and the light chain variable region VL sequence is SEQ ID NO:

5.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody as described in claim 1 or 2.

4. The use of the antibody according to any one of claims 1-2 in the preparation of a product for treating pediatric pneumonia and asthma.

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