Antibodies for the treatment of allergic rhinitis and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an antibody for treating allergic rhinitis and application thereof. The antibody comprises VH CDR1 shown as SEQ ID NO:1, VH CDR2 shown as SEQ ID NO:2, VH CDR3 shown as SEQ ID NO:3, VL CDR1 shown as SEQ ID NO:4, VL CDR2 shown as SEQ ID NO:5 and VL CDR3 shown as SEQ ID NO:6. The application further comprises a nucleic acid molecule encoding the antibody, an expression vector comprising the nucleic acid molecule, a host cell and the use of the host cell in the preparation of a drug for treating or preventing a disease related to IgE-mediated immune response. Animal model experiments show that the antibody of the application can significantly reduce the behavior score of an OVA-induced allergic rhinitis model mouse and the IgE level in serum, and exhibits excellent therapeutic effect, thereby providing a new candidate drug for treating allergic rhinitis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to an antibody for treating allergic rhinitis and its application. Background Technology

[0002] Allergic rhinitis (AR) is a non-infectious chronic inflammatory disease of the nasal mucosa mediated by immunoglobulin E (IgE), triggered by inhaled allergens such as pollen, dust mites, and animal dander. Typical symptoms include paroxysmal sneezing, clear nasal discharge, nasal itching, and nasal congestion, severely impacting patients' quality of life, work efficiency, and sleep, and potentially triggering or exacerbating complications such as asthma, sinusitis, and otitis media. Globally, the incidence of allergic rhinitis is rising annually, becoming a significant public health issue.

[0003] Currently, the treatment of allergic rhinitis mainly follows the principle of "prevention and treatment combined, a four-pronged approach," including avoiding allergens, drug therapy, immunotherapy, and health education. Drug therapy is the primary means of controlling symptoms, and commonly used medications include: second-generation oral antihistamines (such as loratadine and cetirizine), nasal corticosteroids (such as mometasone furoate and fluticasone), and oral leukotriene receptor antagonists (such as montelukast). These medications can effectively relieve symptoms in most patients, but they still have limitations. For example, antihistamines have limited effect on symptoms such as nasal congestion; nasal corticosteroids have a slow onset of action, and some patients are concerned about potential local or systemic side effects (although the risk is very low); long-term use may lead to tolerance or insufficient symptom control. For patients with moderate to severe persistent allergic rhinitis, combination therapy is often required, increasing the complexity of treatment and the economic burden.

[0004] Allergen-specific immunotherapy (AIT, commonly known as "desensitization therapy") is currently the only etiological treatment that may alter the natural course of the disease. However, its treatment course is long, lasting 3-5 years, carries the risk of inducing systemic allergic reactions, and is not effective for all patients. Therefore, there is an urgent clinical need to develop novel, highly effective, and safe therapeutic drugs.

[0005] IgE plays a central role in the pathogenesis of allergic reactions. When an allergen first enters an atopic individual, it stimulates B cells to produce specific IgE against that allergen. IgE binds to high-affinity IgE receptors (FcεRI) on the surface of mast cells and basophils via its Fc fragment, sensitizing the body. When the same allergen re-enters, it cross-links with specific IgE bound to the surface of effector cells, triggering effector cell degranulation and the release of inflammatory mediators such as histamine, leukotrienes, and prostaglandins, leading to an immediate (type I) allergic reaction. In addition, IgE can also participate in antigen presentation through its low-affinity receptor (FcεRII / CD23), exacerbating and prolonging allergic inflammation.

[0006] Based on the above understanding, biologics targeting IgE or its pathway offer a new strategy for the treatment of allergic diseases. Omalizumab, the world's first approved humanized anti-IgE monoclonal antibody, was approved in 2003 for the treatment of moderate to severe allergic asthma, and subsequently expanded to chronic spontaneous urticaria. Omalizumab specifically binds to the Cε3 domain of free IgE, blocking its binding to FcεRI, thereby inhibiting the activation of mast cells and basophils. Clinical trials and real-world studies have confirmed that omalizumab also shows good efficacy in the treatment of allergic rhinitis, especially suitable for moderate to severe patients with severe symptoms and poor control with conventional drug therapy.

[0007] Despite the success of omalizumab, its clinical application still faces several challenges, including: 1) Dosage: It requires subcutaneous injection every 2-4 weeks, demanding high patient compliance; 2) Treatment cost: Its relatively high price limits its accessibility in certain regions and populations; 3) Efficacy variability: It is not effective for all patients, exhibiting individual differences; 4) Safety considerations: Although generally safe, the risk of rare allergic reactions still needs to be monitored. Therefore, developing novel anti-IgE antibodies with novel structures that may possess better efficacy, superior pharmacokinetic properties (such as longer half-life and lower dosing frequency), or better safety has significant clinical and market value.

[0008] The purpose of this invention is to provide a novel monoclonal antibody that can efficiently bind to and neutralize human IgE. This antibody has demonstrated significant therapeutic activity in animal models, improving symptoms of allergic rhinitis and reducing serum IgE levels, and shows promise as a new drug candidate for treating IgE-mediated allergic diseases such as allergic rhinitis. Summary of the Invention

[0009] This invention first provides an antibody for treating allergic rhinitis, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes: (a1) Complementary determination region CDR-H1 as shown in SEQ ID NO:1; (a2) The complementary determination region CDR-H2 as shown in SEQ ID NO:2; and (a3) The complementary determination region CDR-H3 as shown in SEQ ID NO:3; The light chain variable region includes: (b1) Complementary determination region CDR-L1 as shown in SEQ ID NO:4; (b2) The complementary determination region CDR-L2 as shown in SEQ ID NO:5; and (b3) Complementary determination region CDR-L3 as shown in SEQ ID NO:6.

[0010] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8, and / or the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7.

[0011] In some embodiments, the antibody is a full-length IgG antibody, preferably a humanized or fully human IgG antibody.

[0012] The present invention also provides an isolated nucleic acid molecule that encodes the heavy chain variable region, light chain variable region, heavy chain, or light chain of the antibody described above.

[0013] The present invention also provides a recombinant expression vector, characterized in that it comprises the above-mentioned nucleic acid molecules.

[0014] The present invention also provides a host cell, characterized in that it comprises the above-described nucleic acid molecule or the recombinant expression vector as described in claim 5.

[0015] The present invention also provides a method for preparing the above-described antibody, comprising culturing the above-described host cells under conditions that allow antibody expression, and recovering the antibody from the culture.

[0016] The present invention also provides a pharmaceutical composition comprising the antibody described above, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0017] The present invention also provides the use of the above-described antibody or pharmaceutical composition in the preparation of a medicament for treating or preventing IgE-related diseases.

[0018] In some embodiments, the IgE-related diseases are allergic rhinitis, allergic asthma, atopic dermatitis, food allergy, allergic conjunctivitis, or chronic spontaneous urticaria.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects: (1) High specificity and affinity: This invention obtained a monoclonal antibody, AbaE, that can efficiently and specifically bind to human IgE by immunizing fully human antibody mice, cell fusion, screening, and monoclonalization. The results of Examples 1-3 show that the antibody has a high serum titer and good antigen-binding activity.

[0020] (2) Well-defined structure and controllable quality: This invention successfully cloned and determined the complete amino acid sequence (including the CDR region and the framework region) of the variable region of the light and heavy chains of the AbaE antibody, laying the molecular foundation for its large-scale, high-quality industrial production (e.g., expression in CHO cells via recombinant DNA technology). The SDS-PAGE results of Example 4 showed that the purified antibody had the correct molecular weight, high purity, and no significant polymerization or degradation, indicating that it has good stability.

[0021] (3) Significant in vivo therapeutic effect: This invention verified the therapeutic effect of AbaE in a classic OVA-induced IgE / FcεRI humanized mouse model of allergic rhinitis. The experimental results in Example 5 showed that, compared with the negative control group (saline), intravenous injection of AbaE significantly reduced allergic behavioral scores such as nose scratching and sneezing in the model mice, effectively alleviating rhinitis symptoms. Simultaneously, AbaE treatment significantly reduced the level of human IgE in the peripheral blood of mice, suggesting that it may exert its therapeutic effect by binding to and clearing free IgE, blocking the binding of IgE to effector cell receptors. Its efficacy is comparable to or even better than that of the positive control drug omalizumab.

[0022] (4) Promising application prospects: The antibody of this invention has a well-defined sequence and verified in vivo activity, providing a core material basis and experimental evidence for developing a new biological agent to treat IgE-mediated diseases such as allergic rhinitis. The antibody of this invention can be further optimized for humanization, affinity maturation, and Fc segment modification (to regulate its half-life or effector function) to obtain candidate molecules with better drug properties. Attached Figure Description

[0023] Figure 1 The image shows the ELISA results of anti-human IgE antibody titer in mouse serum after immunization.

[0024] Figure 2 The image shows the results of ELISA screening for specific antibodies in hybridoma cell supernatant.

[0025] Figure 3 This is a graph showing the results of ELISA detection of antibody activity in monoclonal hybridoma cell supernatant.

[0026] Figure 4 This is an SDS-PAGE image of the purified monoclonal antibody AbaE. M represents the protein marker, lane 1 is the non-reduced sample, and lane 2 is the reduced sample.

[0027] Figure 5 The figure shows the effect of AbaE on the behavioral scores of mice with OVA-induced allergic rhinitis (**p<0.01 compared with the negative control group).

[0028] Figure 6 The figure shows the effect of AbaE on the serum level of human IgE in mice with OVA-induced allergic rhinitis (**p<0.01 compared with the negative control group). Detailed Implementation

[0029] 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.

[0030] Example 1: Animal Immunization We purchased human IgE from ACROBiosystems, catalog number IGE-H52H9. This protein is expressed by HEK293 and has the sequence P01854-1(106-427) with a His tag at the C-terminus.

[0031] Six-week-old fully human HUGO-Mab mice from Cyagen Biosciences Co., Ltd. were purchased. After acclimatizing to the rearing environment for seven days, the mice were immunized with the aforementioned human IgE protein. The specific procedure was as follows: 50 μg of IgE protein was dissolved in physiological saline, and the concentration was adjusted to 0.5 mg / mL. An equal volume of complete Freund's adjuvant was added dropwise, and emulsification was performed repeatedly using a double syringe until a completely emulsified emulsion was obtained. A drop of the emulsion was placed on the water surface; the drop should not spread rapidly. The mice were carefully removed from their cages, avoiding stress, and the skin at the immunization site on the back was gently lifted. The emulsified antigen was injected. For the first immunization, four injection sites were selected: subcutaneous on the back and neck, with a volume of 50 μL injected at each site. Fourteen days after the first immunization, 50 μg of IgE protein was mixed again with an equal volume of incomplete Freund's adjuvant and emulsified, then injected subcutaneously for a second immunization. Three injection sites were selected for the second immunization. A third immunization was performed 28 days after the first immunization, using the same method as the second immunization. Thirty-eight days after the initial immunization, a small amount of blood was collected from the tail vein of mice, serum was separated, and antibody titers were determined by ELISA. The antibody titer detection method was as follows: 0.05M CBS (pH=9.6) was prepared as the coating buffer. IgE protein was diluted to 1 μg / mL with the coating buffer, and 100 μL / well was added to the microplate. The plate was incubated on a horizontal shaker at room temperature for 5 min, and then incubated overnight at 4°C. The coating buffer was discarded the next day, and 300 μL / well of PBST was added for washing. This process was repeated three times, and the liquid in the wells was thoroughly discarded. 200 μL of PBS containing 1% BSA was added to each well, and the plate was blocked at 37°C for 1.5 h. After discarding the blocking solution, 100 μL / well of sample was added. The serum to be tested was serially diluted with PBS containing 1% BSA, with the lowest dilution being 1:200, followed by 2-fold dilutions, and the highest dilution being 1:102400. The negative control was serum from unimmunized HUGO-Mab mice, and the blank control was a serum-free dilution. After incubation at 37°C for 1 hour, the samples were discarded, and the wells were washed three times with PBST (300 μL / well each time). A 1:5000 dilution of HRP-anti-human IgG antibody was added, and the mixture was incubated at 37°C for 1 hour. Color development was performed using TMB, and the OD650 was read on a microplate reader. The cutoff value was set at 2.1 times the OD value of the negative control; the highest dilution exceeding the cutoff value was considered the serum titer. Experimental results are as follows: Figure 1 As shown, 1, 2, and 3 represent the numbers of different mice, and the horizontal axis represents the dilution factor of the serum to be tested, expressed as Log. The experimental results show that mouse number 1 had the highest antibody titer, with a serum titer of 51200.

[0032] Example 2 Cell Fusion Mice were euthanized by cervical dislocation, and their entire bodies were disinfected by spraying with 75% ethanol. The mice were placed supine on a sterile dissection table, and the spleen was dissected and removed. The spleen was rinsed with sterile PBS, and the surface liquid was blotted dry with sterile filter paper. The spleen was transferred to a new culture dish containing 2 mL of pre-chilled basal culture medium. The spleen was gently ground with the smooth end of a sterile syringe plunger until the spleen tissue was completely dispersed and the culture medium became turbid. A 100-mesh nylon filter was placed on a 50 mL centrifuge tube, and the ground cell suspension was slowly added dropwise to the filter using a pipette. The filter was rinsed 2-3 times with 1 mL of basal culture medium to remove any unground tissue fragments, yielding a preliminary single-cell suspension. The filtered single-cell suspension was centrifuged at 4°C and 300 g for 5 minutes, and the supernatant was discarded. 3-5 mL of pre-chilled erythrocyte lysis buffer was added to the centrifuge tube, and the cell pellet was gently agitated. The mixture was incubated at room temperature for 2-3 minutes, and 10 mL of pre-chilled basal culture medium was added to terminate the lysis. The mixture was then centrifuged at 4°C and 300 g for 5 minutes, and the supernatant was discarded. Add 10 mL of PBS to the cell pellet, gently pipette to resuspend, centrifuge at 300 g for 5 minutes at 4 °C, discard the supernatant, and wash once more. Finally, resuspend the cells in 5 mL of basal culture medium, mix 10 μL of the cell suspension with 10 μL of trypan blue staining solution, add to a hemocytometer, and count the viable cells under a microscope. The viability rate should be ≥90%.

[0033] Mouse myeloma cells SP2 / 0 were resuscitated (IMDM medium + 10% FBS) and cultured to the logarithmic growth phase. SP2 / 0 cells were mixed with isolated spleen cells at a ratio of 1:10 and centrifuged. The supernatant was discarded, and the tube was gently tapped to loosen the pellet. The bottom of the centrifuge tube was immersed in a 30°C water bath, and 1 ml of preheated 50% PEG1500 was added dropwise while gently rotating the tube, completing the addition within 90 seconds. After standing for 90 seconds, 10 ml of serum-free DMEM was added dropwise, and the tube was centrifuged again. The supernatant was discarded, and the cells were resuspended in HAT selective medium containing 20% ​​fetal bovine serum. The cell suspension was diluted to a total volume of 110 mL, and 100 μL / well was added to a 96-well plate and cultured at 37°C with 5% CO2. On day 3, 50 μL of HAT medium was added per well. On day 5, the medium was changed to HT medium, and after 14 days, it was changed to RPMI 1640 basal medium. When hybridoma cells covered 10% of the well bottom area, the supernatant was collected and used for specific screening by ELISA. The experimental method was consistent with the serum titer assay. The antigen used was human IgE protein, and the secondary antibody was HRP-anti-human IgG antibody. The experimental results are as follows: Figure 2 As shown, the horizontal axis represents the dilution factor of the supernatant, expressed as log. The results indicate that fused cell 1 has the best IgE affinity.

[0034] Example 3 Monoclonalization of hybrid cells Hybridoma cells #1 were collected, and monoclonal antibodies were obtained using the limiting dilution method. Hybridoma cells #1 were gently pipetted from the wells of a 96-well plate. After cell counting, the cells were diluted with basal medium, and 0.5 cells / well were added to the 96-well plate. Monoclonal antibody formation was observed after incubation at 37°C and 5% CO2 for approximately 10 days. The supernatant from the wells containing the monoclonal cells was used to detect antibody activity using an ELISA method, following the same procedure as in Example 1. A blank culture medium was used as the negative control. Results are as follows: Figure 3 As shown, the horizontal axis represents the dilution factor of the supernatant, expressed as Log. Monoclonal A showed the best antigen-binding activity, so monoclonal A was expanded and continuously passaged. Cells were cryopreserved after good stability during continuous passage.

[0035] Example 2 Purification of Monoclonal Antibodies Expand monoclonal A cells to two T75 flasks. When cell confluence reaches 80%, discard the culture medium and add 10 mL of fresh complete culture medium, continuing incubation for 48 h. Collect all supernatant, centrifuge at 3000 rpm for 10 min, and collect the supernatant in a new 50 mL centrifuge tube. Filter using a 0.22 µm filter membrane, measure the pH after filtration, and adjust the pH to 7.4 using neutralization buffer. Add 500 µL of Protein A magnetic beads to the supernatant, mix thoroughly, and incubate overnight at 4 °C. The next day, centrifuge at 1500 rpm for 5 min at 4 °C and discard the supernatant. Resuspend the magnetic beads in 1 mL of cold PBS and transfer to a chromatography column. Wash the column with 10 mL of PBS, repeating three times. Add 3 mL of elution buffer to the column, discard the residual PBS, and stopper the bottom of the column. Incubate for 5-10 min. Repeat elution once more after the elution buffer has flowed out. Add 600 µL of neutralization buffer to the collected eluent and mix thoroughly. Transfer the eluent to a 30 kDa ultrafiltration tube and centrifuge at 4000 rpm and 4°C to concentrate the protein solution. After the protein solution volume is reduced to 600 µL, add 5 ml of PBS and centrifuge again under the same conditions. Repeat this process three times until the buffer is completely replaced with PBS. Determine the protein concentration using a spectrophotometer and name the protein AbaE. Take 3 µg of protein, add it to a loading buffer containing DTT, and boil to prepare sample 1. Take another 3 µg of protein, add it to a loading buffer without DTT to prepare sample 2. Perform SDS-PAGE analysis on the samples. The results are as follows: Figure 4As shown in the diagram. M represents the protein marker, lane 1 is the reduced state, and lane 2 is the non-reduced state. The molecular weight of AbaE protein after preliminary purification with Protein G is approximately 150 kDa, consistent with the theoretical molecular weight of IgG. No aggregate bands were observed in the non-reduced state, nor were there any obvious light / heavy chain mismatches or broken-chain bands. Lane 1 represents sample 1, and lane 2 represents sample 2. M represents the protein marker, and sample 1 shows single-molecule heavy and light chain bands without obvious impurities.

[0036] Example 4: Obtaining the variable region sequence of a monoclonal antibody Candidate hybridoma clones were lysed with Trizol and total RNA was extracted. The specific steps were as follows: Cell A was digested and resuspended in DPBS to a concentration of 5*10⁻⁶. 7 Transfer the sample to a 1.5 ml centrifuge tube, centrifuge at 4000 rpm, and discard the supernatant. Add 1 ml Trizol, mix well, and incubate at room temperature for 5 min. Add 0.2 ml chloroform, vortex for 15 s, and incubate for 2 min. Centrifuge at 4°C, 12000 g, for 15 min, and collect the supernatant. Add 0.5 ml pre-chilled isopropanol, gently mix the liquid in the tube, and incubate at room temperature for 10 min. Centrifuge at 4°C, 12000 rpm, for 10 min, and discard the supernatant. Add 1 ml 75% ethanol and gently wash the precipitate. Centrifuge at 4°C, 12000 rpm, for 5 min, and discard the supernatant. Air dry, and dissolve in an appropriate amount of DEPC H2O at 65°C. The total RNA obtained in the above steps was used as a template to synthesize first-strand cDNA. Then, using the first-strand cDNA as a template, subsequent PCR amplification was performed with antibody variable region-specific primers to obtain the nucleic acids of the antibody light and heavy chain variable regions corresponding to hybridoma cells. Agarose gel electrophoresis was performed, and after gel extraction and recovery, Sanger sequencing was conducted to obtain the antibody variable region sequence. The sequences of the antibody heavy chain variable region and light chain variable region are shown in Table 1.

[0037] Table 1. Amino acid sequences of monoclonal antibodies

[0038] Example 5: Evaluation of the therapeutic effect of AbaE in an OVA-induced mouse model of allergic rhinitis Fifteen healthy, active 8-week-old female IgE / FcεRI humanized mice were used. 20 μg of OVA (ovalbumin) was dissolved in 200 μL of physiological saline and thoroughly mixed with 2 mg of aluminum hydroxide gel. The mixture was injected intraperitoneally on days 0, 7, and 14. Local nasal provocation was performed from day 21 to day 27 using 20 μL of physiological saline containing 1% OVA, administered as nasal drops once daily.

[0039] On Day 28, mice were randomly divided into groups of 5 (see Table 2). On Days 28 / 30 / 32 / 34, mice were treated with intravenous injection of 100 μL each time. On Day 49, mice were observed and their behavior was assessed using the following criteria: 0 - no symptoms; 1 - mild nose scratching; 2 - frequent nose scratching; 3 - continuous sneezing and nose scratching; 4 - severe sneezing, runny nose, and wheezing. The behavioral assessment results are shown below. Figure 5 Compared with the negative control group, the AbaE treatment group effectively reduced the behavioral scores of mice and improved the symptoms of allergic rhinitis. Peripheral blood was collected from mice on the same day, and serum IgE levels were measured. The results are as follows: Figure 6 AbaE can reduce peripheral blood IgE levels in mice. (One-way ANOVA, p<0.01) Table 2. Grouping of the allergic rhinitis mouse model A physiological saline negative control group - 100μL B Omalizumab Positive control group 40 mg / kg, 100 μL C AbaE experimental group 100 mg / kg, 100 μL 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 antibody for treating allergic rhinitis, characterized in that, It includes a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes: (a1) Complementary determination region CDR-H1 as shown in SEQ ID NO:1; (a2) The complementary determination region CDR-H2 as shown in SEQ ID NO:2; and (a3) The complementary determination region CDR-H3 as shown in SEQ ID NO:3; The light chain variable region includes: (b1) Complementary determination region CDR-L1 as shown in SEQ ID NO:4; (b2) The complementary determination region CDR-L2 as shown in SEQ ID NO:5; and (b3) Complementary determination region CDR-L3 as shown in SEQ ID NO:

6.

2. The antibody according to claim 1, characterized in that, The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:8, and / or the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

7.

3. The antibody according to claim 1 or 2, characterized in that, The antibody is a full-length IgG antibody, preferably a humanized or fully human IgG antibody.

4. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the heavy chain variable region, light chain variable region, heavy chain, or light chain of the antibody according to any one of claims 1-3.

5. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, It comprises the nucleic acid molecule of claim 4 or the recombinant expression vector of claim 5.

7. A method for preparing the antibody according to any one of claims 1-3, characterized in that, This includes culturing the host cells of claim 6 under conditions that allow antibody expression, and recovering the antibody from the culture.

8. A pharmaceutical composition, characterized in that, It comprises the antibody as described in any one of claims 1-3, and a pharmaceutically acceptable carrier, diluent, or excipient.

9. Use of the antibody according to any one of claims 1-3 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for the treatment or prevention of IgE-related diseases.

10. The use according to claim 9, characterized in that, The diseases associated with IgE are allergic rhinitis, allergic asthma, atopic dermatitis, food allergy, allergic conjunctivitis, or chronic spontaneous urticaria.

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