Anti-human interleukin-5 nanoantibody and its application

Abstract

The present invention belongs to the field of medicine and discloses an anti-human interleukin-5 nanobody and its application. The amino acid sequences of the three complementary determining regions (CDR1, CDR2, and CDR3) of the anti-IL-5 antibody are IDAMG, AISMTGYSTYYAESMKG, and DAYGDYGIGPYLEV, respectively. The IL-5 nanobody of the present invention can bind to the hIL-5 antigen with high specificity, can block the binding of IL-5 to IL-5Rα, and has human-monkey cross-activity. It is expected to be developed into a new type of targeted IL-5 inhalation preparation, filling the market gap of targeted IL-5 inhalation administration and benefiting more patients.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to anti-human interleukin 5 (hIL-5) nanoantibodies and applications thereof. Background Art

[0002] Severe eosinophilic asthma (SEA) is a difficult-to-treat respiratory disease with poor clinical control. Eosinophils play a crucial role in the pathogenesis of SEA, causing airway epithelial damage and bronchial remodeling. Eosinophils are associated with a higher frequency of exacerbations, which in turn reduces lung function. The current treatment for asthma is inhaled corticosteroids, but long-term use can lead to osteoporosis and hypertension. Biologic agents have shown promising results in treating severe asthma. Type 2 inflammation occurs in 50% to 70% of asthma patients. The production of Th2-mediated inflammatory factors, such as IL-5, IL-4, and IL-13, increases eosinophils, IgE, and exhaled nitric oxide (FENO). Therefore, reducing eosinophil counts by inhibiting proinflammatory cytokines is a highly beneficial therapeutic option for treating SEA.

[0003] Human interleukin-5 (IL-5) is a homodimeric cytokine linked by interchain disulfide bonds. It is crucial for regulating the proliferation, activation, and maturation of eosinophils, promoting their release from the bone marrow into the circulation. Although Th2 lymphocytes are the primary source of IL-5 production, mast cells, group II innate lymphoid cells (ILC2s), natural killer T cells, and even eosinophils also express and produce IL-5. IL-5Rα is an IL-5 binding receptor expressed on the surface of mature eosinophils and basophils. The intermediate complex formed by IL-5 binding to IL-5Rα recruits the β common (βc) receptor (CD131) to form a ternary complex, which then activates the JAK / STAT signaling cascade, promoting eosinophil proliferation. Currently available anti-IL-5 antibodies include mepolizumab and reslizumab. They neutralize IL-5 levels in the body, thereby reducing eosinophil levels, inhibiting eosinophil differentiation and activation, and ultimately slowing asthma exacerbations. In clinical trials, antibodies targeting the IL-5 / IL-5Rα complex have shown promise in reducing the rate of severe asthma exacerbations, improving forced expiratory volume in 1 second (FEV1), and reducing the need for oral glucocorticoids. Furthermore, since eosinophils are involved in mucus formation, reducing the number of airway eosinophils can reduce airway obstruction and improve lung function. Anti-IL-5 antibodies have been shown to be effective as an emergency treatment for steroid-refractory acute severe eosinophilic asthma. Currently available anti-IL-5 monoclonal antibodies require repeated injections, significantly impacting patient compliance. Furthermore, injections require high doses, resulting in high systemic exposure and an increased risk of side effects. Therefore, inhaled administration of the antibody may be an alternative approach.

[0004] The efficiency of inhaled antibody drugs is negatively correlated with the antibody's molecular weight. Before reaching the alveoli, the airways branch approximately 16 to 17 times. Particle deposition is primarily influenced by particle size, weight, and respiratory airflow. Particles larger than 5 μm remain primarily in the upper respiratory tract, such as the throat and airways, due to gravity and sedimentation, and are difficult to reach deep into the lungs. Particles smaller than 0.5 μm are easily exhaled. Drugs with particle sizes between 1 and 5 μm can diffuse into the lower respiratory tract via Brownian motion and are therefore most suitable for inhaled administration. Because traditional monoclonal antibodies have a large molecular weight (approximately 150 kDa), the particle diameter after aerosolization is greater than 5 μm, resulting in low absorption rates in lung tissue. Nanobodies (Nb) are the variable regions of heavy-chain antibodies (HcAbs) naturally lacking light chains, discovered in camelids by Belgian scientist Hamers-Casterman's laboratory in 1989. Nanobody crystals are 2.5 nm in diameter and 4 nm long, with a molecular weight of approximately 12-15 kDa, just one-tenth the molecular weight of traditional antibodies. They are the smallest known natural antibodies that bind to their target antigens. Structurally distinct from traditional antibodies, nanobodies offer advantages such as small molecular weight, excellent tissue penetration, simple structure facilitating molecular design, low immunogenicity, and ease of humanization, as well as high affinity, good solubility, high expression levels, excellent heat and low pH stability, and ease of expression, purification, and microbial expression, making them ideal for the development of inhaled antibody drugs.

[0005] Therefore, the development of a new anti-IL-5 nanoantibody for inhaled administration has good clinical application prospects and is expected to fill the market gap in targeted IL-5 inhaled administration and benefit more patients. Summary of the Invention

[0006] The object of the present invention is to overcome at least one deficiency of the prior art and to provide an anti-human interleukin-5 nanobody and its application.

[0007] The technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides:

[0009] An anti-human interleukin-5 nanobody, the amino acid sequences of its three complementary determining regions CDR1, CDR2, and CDR3 are IDAMG, AISMTGYSTYYAESMKG, and DAYGDYGIGPYLEV, respectively.

[0010] In some examples of anti-human interleukin 5 nanobodies, four framework regions are included, FR1, FR2, FR3 and FR4, and the amino acid sequences of FR1, FR2, FR3 and FR4 are EVQVVESGGGLVQPGGSLRLSCAASTSIFS, WYRQAPGKGLEWVS, RFTISRDNAKNTVYLQMNSLKPEDTAVYYCHA and WGQGTLVTVSS, respectively.

[0011] In some examples of anti-human interleukin 5 nanobody, its amino acid sequence is: EVQVVESGGGLVQPGGSLRLSCAASTSIFSIDAMGWYRQAPGKGLEWVSAISMTGYSTYYAESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCHADAYGDYGIGPYLEVWGQGTLVTVSS.

[0012] The second aspect of the present invention provides:

[0013] A gene encoding the anti-human interleukin-5 nanobody according to the first aspect of the present invention.

[0014] Specifically, the specific gene sequence can be codon-optimized according to different expression systems to obtain better expression results.

[0015] The third aspect of the present invention provides:

[0016] An expression system for expressing the anti-human interleukin-5 nanobody described in the first aspect of the present invention.

[0017] A fourth aspect of the present invention provides:

[0018] A preparation targeting human interleukin 5, comprising the anti-human interleukin 5 nanobody according to the first aspect of the invention.

[0019] In some examples of agents, it is a detection reagent.

[0020] In some examples of the preparation, it is a drug for treating human interleukin-5 related diseases.

[0021] In some examples of the preparation, the preparation is a drug for treating human interleukin-5 related diseases, and the human interleukin-5 related diseases are selected from asthma and chronic obstructive pulmonary disease.

[0022] In some examples of the preparation, it is an inhalant, an injection, or a transdermal absorption preparation.

[0023] A fifth aspect of the present invention provides:

[0024] The anti-human interleukin-5 nanobody described in the first aspect of the present invention is used in the preparation of a human interleukin-5 targeted preparation.

[0025] The beneficial effects of the present invention are:

[0026] The IL-5 nanoantibody of the present invention can bind to the hIL-5 antigen with high specificity, can block the binding of IL-5 and IL-5Rα and has human-monkey cross-activity. It is expected to be developed into a new type of targeted IL-5 inhalation preparation, filling the market gap of targeted IL-5 inhalation administration and benefiting more patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figures 1 to 4 This is the test result of the binding of recombinant nanoantibody to human IL-5.

[0028] Figures 5 to 8 This is the test result of the binding of recombinant nanobody to monkey IL-5.

[0029] Figures 9-10 This is the ELISA blocking test result of the recombinant nanobody.

[0030] Figure 11 This is the FACS blocking test result of the recombinant nanobody AIL-A96-Fc antibody.

[0031] Figure 12 This is the result of the cell proliferation inhibition test of the recombinant nanobody AIL-A96-Fc antibody. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further illustrated below in conjunction with experiments.

[0033] Example 1: Natural library screening

[0034] Phage suspensions prepared from a preserved alpaca natural library (reservoir capacity 2.20E+13 cfu and 3.30E+13 cfu) were diluted with 2.5% BSA and blocked for negative screening. Phages were collected after negative screening incubation. IL-5-specific nanobodies were screened using phage display technology. The specific screening method is described in Example 3 of patent CN110144011B. After three rounds of "bind-wash-elute" enrichment, the final enrichment is shown in Table 1. The pools were screened and tested, and single clones from the pool with good antigen binding were selected for initial Phage ELISA screening. Screening was performed using hIL-5-cHis antigen-coated plates. A total of 461 positive clones were obtained, 50 of which were sequence-unique molecules. 30 molecules were ultimately selected for recombinant expression.

[0035] Table 1: Alpaca Natural Library Selection Data

[0036] serial number Screening antigens Antigen concentration Negative sieve Input Input(cfu) Output(cfu) Blank(cfu) OP / IP‰ OP / B 1-1 IL-5-His 100 μg / mL N / A Alpaca Natural Library 2.20E+13 1.40E+08 N / A 0.01 N / A 1-2 IL-5-His-bio 300 nM N / A Alpaca Natural Library 3.30E+13 3.60E+08 N / A 0.01 N / A 2-1 IL-5-His 30 μg / mL N / A 1-1 1.80E+12 1.00E+07 1.12E+07 0.01 0.89 2-2 IL-5-His-bio 100 nM N / A 1-1 9.00E+11 4.20E+06 4.80E+07 0 0.09 2-3 IL-5-Fc 30 μg / mL BCMA 1-1 1.20E+12 3.20E+07 1.12E+07 0.03 2.86 2-4 IL-5-Fc-bio 100 nM BCMA 1-1 5.00E+11 4.80E+06 4.80E+07 0.01 0.1 2-5 IL-5-His 30 μg / mL N / A 1-2 1.40E+12 6.40E+06 1.12E+07 0 0.57 2-6 IL-5-His-bio 100 nM N / A 1-2 6.50E+11 1.80E+07 4.80E+07 0.03 0.38 2-7 IL-5-Fc-bio 100 nM BCMA 1-2 6.00E+11 2.10E+07 4.80E+07 0.04 0.44 2-8 IL-5-Fc 30 μg / mL BCMA 1-2 1.00E+12 1.12E+07 1.12E+07 0.01 1 3-1 IL-5-His 10 μg / mL N / A 2-1 4.60E+11 2.40E+06 6.00E+06 0.01 0.4 3-2 IL-5-His 10 μg / mL N / A 2-2 2.40E+11 3.20E+06 6.00E+06 0.01 0.53 3-3 IL-5-His 10 μg / mL N / A 2-3 3.60E+11 5.20E+06 6.00E+06 0.01 0.87 3-4 IL-5-His 10 μg / mL N / A 2-4 5.00E+11 8.00E+06 6.00E+06 0.02 1.33 3-5 IL-5-His-bio 30 nM N / A 2-5 2.40E+11 4.40E+07 9.20E+08 0.18 0.05 3-6 IL-5-His-bio 30 nM N / A 2-6 1.70E+11 8.00E+08 9.20E+08 4.71 0.87 3-7 IL-5-His-bio 30 nM N / A 2-7 1.15E+11 1.72E+08 9.20E+08 1.5 0.19 3-8 IL-5-His-bio 30 nM N / A 2-8 1.35E+11 5.60E+07 9.20E+08 0.41 0.06

[0037] Example 2: In vitro recombinant expression and purification of nanobodies and detection of binding activity to human IL-5 protein

[0038] 30 nanoantibodies fused with Fc tags were recombinantly expressed in CHO cells and purified using protein A before ELISA detection. Human IL-5 was diluted to 2 μg / mL with PBS and added to the enzyme-labeled plate, 30 μL per well, coated overnight, washed 5 times with PBST, blocked with 5% milk at 37°C for 1 hour, added with gradient dilutions of recombinant antibodies, incubated at 37°C for 1 hour, washed 5 times with PBST, and then added with secondary antibody Anti-human-IgG-Fc-HRP (diluted with PBS 1:10000) and incubated at 37°C for 1 hour, then washed 5 times with PBST, 30 μL of TMB color development solution was added to each well, reacted at room temperature in the dark for 5 minutes, and then 2M sulfuric acid was added to terminate the reaction. The absorbance value was measured at 450nm by a microplate reader. The results are as follows: Figures 1 to 4 As shown, AIL-A12-Fc, AIL-A14-Fc, AIL-A23-Fc, AIL-B127-Fc, AIL-A76-Fc, AIL-B90-Fc, AIL-A96-Fc, and AIL-B124-Fc have good binding to human IL-5.

[0039] Example 3: Detection of binding activity of recombinant nanobody to monkey IL-5 protein

[0040] Monkey IL-5 was diluted to 2 μg / mL with PBS and added to the enzyme-labeled plate, 30 μL per well, coated overnight, washed 5 times with PBST, blocked with 5% milk at 37°C for 1 h, added with gradient dilutions of recombinant antibodies, incubated at 37°C for 1 h, washed 5 times with PBST, added with secondary antibody Anti-human-IgG-Fc-HRP (diluted with PBS 1:10000) and incubated at 37°C for 1 h, then washed 5 times with PBST, added 30 μL TMB colorimetric solution to each well, reacted at room temperature in the dark for 5 min, and then added 50 μL 2M sulfuric acid to terminate the reaction. The absorbance value was measured at 450 nm with an enzyme-labeled instrument. The results are as follows: Figures 5 to 8 As shown, AIL-A12-Fc, AIL-A14-Fc, AIL-A23-Fc, AIL-B127-Fc, AIL-B124-Fc, AIL-A76-Fc, AIL-A96-Fc, and AIL-B90-Fc have better binding to monkey IL-5.

[0041] Example 4: Recombinant Nanobody ELISA Blocking Detection

[0042] Dilute hIL-5-hFc to 2 μg / mL with PBS, take 30 μL and add it to the enzyme-labeled plate, and incubate it at 4°C overnight. Wash the plate three times with PBST and block it with 5% milk at room temperature for 2 hours. Wash the plate three times with PBST, dilute the recombinant nanoantibody to be tested with 1% milk, add it to the enzyme-labeled plate, 30 μL per well, and incubate at room temperature for 1 hour. Add hIL-5Rα-cHis (4 μg / mL) without washing the plate and incubate it at room temperature for 1 hour. Wash the plate three times with PBST, add 6×His-HRP (proteintech; HRP-66005) antibody diluted 1:4000, and incubate it at room temperature for 1 hour. Add 30 μL of TMB color development solution to each well, react at room temperature in the dark for 5 minutes, then add 50 μL of 2M sulfuric acid to terminate the reaction, and measure the absorbance at 450 nm with an enzyme reader. The results are as follows: Figure 9 As shown, AIL-A96-Fc has a good ELISA blocking effect.

[0043] After sequencing, the amino acid sequence of AIL-A96-Fc was EVQVVESGGGLVQPGGSLRLSCAASTSIFSIDAMGWYRQAPGKGLEWVSAISMTGYSTYYAESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCHADAYGDYGIGPYLEVWGQGTLVTVSS (SEQ ID NO.: 8), wherein the amino acid sequences of the three complementary determining regions CDR1, CDR2, and CDR3 were IDAMG (SEQ ID NO.: 1), AISMTGYSTYYAESMKG (SEQ ID NO.: 2), and DAYGDYGIGPYLEV (SEQ ID NO.: 3), respectively; the amino acid sequences of the four framework regions FR1, FR2, FR3, and FR4 were EVQVVESGGGLVQPGGSLRLSCAASTSIFS (SEQ ID NO.: 4), WYRQAPGKGLEWVS (SEQ ID NO.: 5), respectively. NO.: 5), RFTISRDNAKNTVYLQMNSLKPEDTAVYYCHA (SEQ ID NO.: 6) and WGQGTLVTVSS (SEQ ID NO.: 7).

[0044] Example 5: Flow cytometry detection of the blocking effect of recombinant nanobodies

[0045] A stable cell line co-expressing IL-5Rα and CD131 proteins was constructed. The cells were collected by centrifugation at 1000 rpm for 5 min, washed once with 30 mL of PBS, and then resuspended in FACS staining buffer to prepare a cell suspension. The cell density was adjusted to 1 × 10 6cells / mL, add 100 μL per well to a 96-well plate, centrifuge, and discard the supernatant. Dilute the antibody with FACS staining buffer to a concentration of 20.00000, 2.00000, 1.00000, 0.5000, 0.25000, 0.12500, 0.06250, and 0.00625 μg / mL, for a total of 8 dilutions. Dilute the ligand hIL-5-hFc protein with FACS buffer to a concentration of 0.2 μg / mL. Antibody and ligand were mixed at a 1:1 ratio and incubated at 4°C for 1 hour. The ligand concentration in the system was 0.1 μg / mL, and the antibody concentrations were 10.00000, 1.00000, 0.50000, 0.25000, 0.12500, 0.06250, 0.03125, and 0.0016 μg / mL. 100 μL of the antibody and hIL-5-hFc incubation solution was added to a 96-well plate containing cells, resuspended, and incubated at 4°C for 60 minutes. After incubation, cells were washed with 200 μL of FACS staining buffer, centrifuged at 1000 rpm for 5 minutes, and the supernatant discarded. A secondary antibody, Goat pAb to huIgG, was diluted 1:300 in FACS staining buffer and 100 μL / well was added to the 96-well plate. The plate was incubated at 4°C for 30 minutes. Wash twice with 200 μL of FACS staining buffer each time, and finally resuspend in 250 μL of FACS staining buffer, transfer to a flow tube, and detect the FITC signal value using a flow cytometer. The results are as follows Figure 10 As shown in Figure 3, AIL-A96-Fc can significantly block the binding of IL-5 to cell surface IL-5Rα at the FACS level.

[0046] Example 6: Proliferation inhibition assay of recombinant nanobody TF-1

[0047] Prepare IL-5-dependent TF-1 cells one day in advance, collect all cells by centrifugation, wash them once with serum-free 1640 medium, and culture them overnight in 10% 1640 medium without GM-CSF. Before the proliferation inhibition assay, collect cells by centrifugation, resuspend them in 5% 1640 medium, and adjust the cell density to 2E5 cells / mL. Add 50 μL of cells to each well of a 96-well plate. Serially dilute the antibody with 5% 1640 medium, and dilute the IL-5 protein to 10 ng / mL with 5% 1640 medium. Mix the diluted antibody and IL-5 protein in a 1:1 ratio and incubate for 30 minutes. Add the incubated solution to a 96-well plate containing A17 cells. After incubating the cell culture plate in a 37°C, 5% CO2 incubator for 48 hours, add 10 μL of CCK-8 and incubate for 30 minutes before measuring the OD value. 450 The value of Figure 11 As shown, AIL-A96-Fc can significantly inhibit the proliferation of TF-1 cell line.

[0048] Based on the above results, AIL-A96-Fc not only has human-monkey cross-binding activity, but also has a good blocking effect, which can block the binding of IL-5 and IL-5Rα. In addition, the nanoantibody has a small molecular weight and good stability, making inhalation administration possible. It is expected to fill the market gap of targeted IL-5 inhalation administration, quickly relieve asthma symptoms, and treat uncontrolled severe asthma.

[0049] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. An anti-human interleukin-5 nanobody, characterized in that The amino acid sequences of its three complementarity determining regions CDR1, CDR2 and CDR3 are IDAMG, AISMTGYSTYYAESMKG and DAYGDYGIGPYLEV, respectively.

2. The anti-human interleukin-5 nanobody according to claim 1, characterized in that It includes four framework regions FR1, FR2, FR3 and FR4. The amino acid sequences of FR1, FR2, FR3 and FR4 are EVQVVESGGGLVQPGGSLRLSCAASTSIFS, WYRQAPGKGLEWVS, RFTISRDNAKNTVYLQMNSLKPEDTAVYYCHA and WGQGTLVTVSS, respectively.

3. The anti-human interleukin-5 nanobody according to claim 1, characterized in that Its amino acid sequence is: EVQVVESGGGLVQPGGSLRLSCAASTSIFSIDAMGWYRQAPGKGLEWVSAISMTGYSTYYAESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCHADAYGDYGIGPYLEVWGQGTLVTVSS.

4. A gene encoding the anti-human interleukin-5 nanobody according to any one of claims 1 to 3.

5. An expression system for expressing the anti-human interleukin-5 nanobody according to any one of claims 1 to 3, comprising the gene according to claim 4.

6. A detection reagent for human interleukin-5, characterized in that Contains the anti-human interleukin-5 nanobody according to any one of claims 1 to 3.

7. Use of the anti-human interleukin-5 nanobody according to any one of claims 1 to 3 in the preparation of a human interleukin-5 detection reagent.

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