IL-5 binding molecules, methods for their preparation and uses

Abstract

An IL-5-binding molecule, its preparation method, and its use are described. The binding molecule comprises the following complementarity-determining regions: a CDR1 amino acid sequence selected from any of the sequences shown in SEQ ID NOs: 43 to 49, a CDR2 amino acid sequence selected from any of the sequences shown in SEQ ID NOs: 50 to 56, and a CDR3 amino acid sequence selected from any of the sequences shown in SEQ ID NOs: 57 to 62. The binding molecule specifically binds to IL-5 and can effectively inhibit IL-5-induced cell proliferation.

Description

[Technical field]

[0001] The present invention relates to the field of medical biotechnology, in particular to binding molecules against IL-5, their preparation methods and their uses. [Background technology]

[0002] Human interleukin 5, also called IL-5, is a cytokine involved in hematopoiesis and inflammation, together with IL-13 and granulocyte-macrophage colony-stimulating factor (GM-CSF). All three cytokines promote the production, function and survival of eosinophils, and thus can affect inflammatory diseases. For example, eosinophils function primarily as effectors in asthma, atopic dermatitis and allergic rhinitis.

[0003] IL-5 has been noted by most researchers as an eosinophil-specific cytokine, with increased IL-5 mRNA and protein levels in lung tissue and bronchoalveolar lavage fluid (BAL) from symptomatic asthma patients. Correlations between IL-5 levels, allergen stimulation, and disease activity have also been observed by researchers. However, it is clear that in addition to IL-5, GM-CSF and IL-3 also play a role in eosinophil production and activation in asthma, and GM-CSF and IL-3 have been shown to be synthesized at sites where allergic inflammation occurs. Expression of these cytokines can help increase the total number of infiltrating eosinophils and the degree of eosinophil activation. It is also possible that these cytokines play a role in eosinophil infiltration at different stages. Recent kinetic data from patients who underwent antigen challenge showed that IL-5 levels increased from days 2 to 7, while GM-CSF levels peaked on day 2 and continued to increase on day 16.

[0004] IL-5, GM-CSF and IL-3 stimulate eosinophils and other normal and cancer cells by binding to cell surface receptors. The cell surface receptors include ligand-specific α and β chains (βc) shared by the three receptors. Binding to the α chain of each receptor is the first step in receptor activation. However, binding to the α chain alone is not sufficient for activation. The ligand then recruits βc, which is followed by a step that has two major functional consequences: first, it makes the binding of IL-5, GM-CSF and IL-3 virtually irreversible, which then leads to full receptor activation. As the main signaling component of these receptors, βc leads to the activation of JAK-2, STAT-5 and other signaling molecules, which ultimately leads to the excessive cellular activity normally associated with IL-5, GM-CSF and IL-3 stimulation, such as eosinophil adhesion, which results in degranulation and cytotoxicity and extended cell survival.

[0005] To inhibit or antagonize the activity of cytokines for eosinophil activation, researchers have tried three main methods. One of these methods uses antibodies against related cytokines. For example, antibodies against IL-5 are used in animal models of allergen-induced asthma. This method has shown a relatively long-acting effect in preventing high responses due to the influx of eosinophils into airways and bronchi. However, there is still a lack of high affinity, IL-5 specific antibodies and drugs that target IL-5. In this respect, the present invention is provided. Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide an IL-5 binding molecule, a method for its preparation and its use. [Means for solving the problem]

[0007] The present invention is realized as follows. According to a first aspect, an embodiment of the present invention provides an IL-5 binding molecule capable of specifically binding to IL-5 and comprising at least one immunoglobulin single variable domain comprising complementarity determining regions CDR1, CDR2 and CDR3.

[0008] The amino acid sequence of CDR1 is selected from any of the sequences shown in SEQ ID NOs: 43 to 49, the amino acid sequence of CDR2 is selected from any of the sequences shown in SEQ ID NOs: 50 to 56, and the amino acid sequence of CDR3 is selected from any of the sequences shown in SEQ ID NOs: 57 to 62.

[0009] According to a second aspect, an embodiment of the invention provides an isolated nucleic acid encoding an IL-5 binding molecule of the previous embodiment.

[0010] According to a third aspect, an embodiment of the present invention provides a recombinant vector comprising the isolated nucleic acid of the previous embodiment.

[0011] According to a fourth aspect, an embodiment of the present invention provides a host cell comprising a recombinant vector of the previous embodiment.

[0012] According to a fifth aspect, an embodiment of the present invention provides a method for preparing an IL-5 binding molecule comprising culturing a host cell of the previous embodiment to obtain the IL-5 binding molecule.

[0013] According to a sixth aspect, an embodiment of the invention provides a conjugate for binding to an IL-5 protein comprising a conjugation moiety and an IL-5 binding molecule of the previous embodiment, A conjugate is provided in which the conjugation moiety is conjugated to the IL-5 binding molecule, and the conjugation moiety includes a marker and / or a compound for detection.

[0014] According to a seventh aspect, an embodiment of the invention provides a kit for detecting IL-5 comprising an IL-5 binding molecule of the previous embodiment.

[0015] According to an eighth aspect, an embodiment of the invention provides the use of an IL-5 binding molecule of a previous embodiment in the preparation of a medicament which targets IL-5 for the treatment of a disease. Effect of the Invention

[0016] The present invention has the following advantageous effects. An embodiment of the present invention provides an IL-5 binding molecule, a method for its preparation and its use. The binding molecule can specifically bind to IL-5 and comprises at least one immunoglobulin single variable region comprising complementarity determining regions CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 is selected from any of the sequences shown in SEQ ID NOs: 43 to 49, the amino acid sequence of CDR2 is selected from any of the sequences shown in SEQ ID NOs: 50 to 56, and the amino acid sequence of CDR3 is selected from any of the sequences shown in SEQ ID NOs: 57 to 62. The binding molecule can specifically bind to IL-5 and effectively inhibit IL-5-induced cell proliferation, and can be used for the prevention, diagnosis and / or treatment of IL-5-related diseases.

[0017] In order to more clearly describe the technical solutions of the embodiments of the present invention, the following briefly describes the accompanying drawings that are required to describe the embodiments. It should be understood that the following accompanying drawings only show some embodiments of the present invention, and these drawings cannot be considered as limiting the scope of the invention. Those skilled in the art can still derive other drawings from the accompanying drawings without creative efforts. [Brief description of the drawings]

[0018] [Figure 1] 1 shows the results of gel electrophoresis analysis of human recombinant IL-5 protein in Example 1. [Diagram 2]Figure 1 shows the enrichment of recombinant IL-5 protein after screening of the nanobody library in Example 1. P / N is the number of monoclonal bacteria grown after infection of TG1 bacteria with phages eluted from positive wells during biopanning / the number of monoclonal bacteria grown after infection of TG1 bacteria with phages eluted from negative wells. This parameter gradually increases during enrichment. I / E is the total amount of phages added to positive wells during biopanning in each round / the total amount of phages eluted from positive wells during biopanning. This parameter gradually approaches 1 during enrichment. [Diagram 3] 1 shows the results of alignment of humanized variants of antibody strains 1B3 and 2B3 in Example 2. [Figure 4] 1 shows the results of analysis of the binding of 12 antibody strains expressed in E. coli obtained in Example 1 to IL-5 in Verification Example 1. [Diagram 5] 1 is a graph showing the dose-response relationship of binding of Tab1 and Tab2 to IL-5 in Verification Example 2. [Figure 6] 1 is a graph showing the dose-response relationship of IL-5-induced TF-1 cell proliferation neutralized with control antibody 1 (Tab1) and control antibody 2 (Tab2) in Verification Example 2. [Figure 7] 1 is a graph showing the dose-response relationship of IL-5-induced TF-1 cell proliferation neutralized with the IL-5-specific Fc-fused single domain antibody obtained in Example 3 in Verification Example 3. [Figure 8] 1 is a graph showing the dose-response relationship of IL-5-induced TF-1 cell proliferation neutralized with different humanized Fc-fused single domain antibodies obtained in Example 3 in Verification Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Unless specific conditions are specified in the examples, they are carried out according to normal conditions or conditions recommended by the manufacturers. All reagents or equipment without manufacturer's names are common products available in the market.

[0020] <Terminology> As used herein, a "single domain antibody" (sdAb), also known as a nanobody, is an antibody that does not naturally contain a light chain and only comprises one variable region of a heavy chain (VHH).

[0021] As used herein, the term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (e.g., an animal antibody) with the constant region of a human antibody, or an antibody obtained by grafting the complementarity determining regions (sequences of CDR1-3) of a target antibody into the variable region of a human antibody, or an antibody obtained by subjecting the target antibody to amino acid mutations in accordance with the characteristics of the framework regions (FR1-4) of a human antibody. Humanized antibodies can be obtained by synthesis or site-directed mutagenesis.

[0022] As used herein, a "bispecific antibody" is a small bivalent bispecific antibody fragment that can simultaneously recognize two antigens. Hollinger et al. linked the gene of the light chain variable region (VLA) of an antibody of antigen A to the heavy chain variable region (VHB) of an antibody of antigen B with a short peptide molecule, and similarly linked VHA to VLB, and introduced the two sets of chimeric genes into a bicistronic expression plasmid to construct an expression plasmid for a bispecific antibody. After expression, VLA-VHB was cross-linked to VHA-VLB to form a bispecific antibody. In the present application, one of the antigens that can be recognized by a bivalent antibody is IL-5 protein. Other antigens that can be recognized by a bivalent antibody can be selected from any of the existing antigens.

[0023] As used herein, a "multivalent antibody", also called a multi-antibody, refers to an antibody (similar to a bispecific antibody) that has an altered structure that can simultaneously recognize various antigens (similar to a bispecific antibody). In the present invention, one of the antigens that can be recognized by a multivalent antibody is the IL-5 protein.

[0024] As used herein, "CDR" refers to the complementarity determining region of an antibody. An antibody typically comprises two variable regions: a heavy chain variable region and a light chain variable region. A heavy chain variable region or a light chain variable region typically comprises three CDRs.

[0025] <Implementation> An embodiment of the present invention provides an IL-5 binding molecule, the IL-5 binding molecule being capable of specifically binding to IL-5 and comprising at least one immunoglobulin single variable domain comprising complementarity determining regions CDR1, CDR2 and CDR3; The amino acid sequence of CDR1 is selected from any of the sequences shown in SEQ ID NOs: 43 to 49, the amino acid sequence of CDR2 is selected from any of the sequences shown in SEQ ID NOs: 50 to 56, and the amino acid sequence of CDR3 is selected from any of the sequences shown in SEQ ID NOs: 57 to 62.

[0026] Preferably, the amino acid sequences of the complementarity determining regions CDR1, CDR2, and CDR3 are any of the following (1) to (13): (1) SEQ ID NOs: 43, 56 and 58, (2) SEQ ID NOs: 49, 51 and 60, (3) SEQ ID NOs: 47, 56 and 57, (4) SEQ ID NOs: 45, 54 and 57, (5) SEQ ID NOs: 46, 50 and 61, (6) SEQ ID NOs: 47, 56 and 62, (7) SEQ ID NOs: 48, 56 and 57, (8) SEQ ID NOs: 45, 53 and 57, (9) SEQ ID NOs: 44, 55 and 57, (10) SEQ ID NOs: 43, 56 and 59, (11) SEQ ID NOs: 43, 52 and 59, (12) SEQ ID NOs: 47, 56, and 59, and (13) SEQ ID NOs: 47, 52, and 59.

[0027] In some embodiments, the immunoglobulin single variable domain further comprises a framework region comprising FR1, FR2, FR3 and FR4. The structure of a single domain antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0028] The amino acid sequence of FR1 is selected from any of the sequences shown in SEQ ID NOs: 63 to 68, the amino acid sequence of FR2 is selected from any of the sequences shown in SEQ ID NOs: 69 to 72, the amino acid sequence of FR3 is selected from any of the sequences shown in SEQ ID NOs: 73 to 86, and the amino acid sequence of FR4 is shown in SEQ ID NO: 87.

[0029] Preferably, the framework regions FR1, FR2, and FR3 of the immunoglobulin single variable region have any of the sequences shown in (14) to (28) below: (14) SEQ ID NOs: 65, 72 and 85, (15) SEQ ID NOs: 65, 72 and 82, (16) SEQ ID NOs: 64, 71 and 86, (17) SEQ ID NOs: 65, 72 and 73, (18) SEQ ID NOs: 65, 72 and 84, (19) SEQ ID NOs: 66, 69 and 83, (20) SEQ ID NOs: 68, 72 and 76, (21) SEQ ID NOs: 66, 69 and 79, (22) SEQ ID NOs: 63, 72 and 85, (23) SEQ ID NOs: 67, 70 and 80, (24) SEQ ID NOs: 65, 72 and 78, (25) SEQ ID NOs: 65, 72 and 77, (26) SEQ ID NOs: 67, 70 and 81, (27) SEQ ID NOs: 65, 72, and 74, and (28) SEQ ID NOs: 65, 72, and 75.

[0030] Preferably, the immunoglobulin single variable domain is a VHH. Preferably, the amino acid sequence of the VHH is selected from any of the sequences shown in SEQ ID NOs: 1 to 12.

[0031] Preferably, said VHH is a humanized VHH. Preferably, the sequence of the humanized VHH is selected from any of the sequences shown in SEQ ID NOs: 13 to 21.

[0032] Preferably, the binding molecule further comprises an immunoglobulin Fc region linked to the VHH. Preferably, the immunoglobulin Fc region is a human immunoglobulin Fc region. Preferably, the human immunoglobulin Fc region is a human IgG4 Fc region.

[0033] An embodiment of the invention provides an isolated nucleic acid encoding an IL-5 binding molecule of any of the above embodiments. Preferably, the nucleic acid sequence is selected from any of the sequences shown in SEQ ID NOs: 22-42.

[0034] An embodiment of the present invention provides a recombinant vector comprising the isolated nucleic acid of any of the above embodiments. In any embodiment, the recombinant vector can be a plasmid, a phage, or a viral vector.

[0035] An embodiment of the present invention provides a host cell comprising the recombinant vector of the previous embodiment. In any embodiment, the host cell can be a prokaryotic or eukaryotic cell.

[0036] An embodiment of the invention provides a method for preparing an IL-5 binding molecule comprising culturing a host cell of the above embodiment to obtain the IL-5 binding molecule.

[0037] The binding molecules described in any of the above embodiments may be prepared synthetically or may be obtained by biological expression following prior synthesis of their encoding genes.

[0038] An embodiment of the invention provides a conjugate for binding to an IL-5 protein comprising a conjugation moiety and an IL-5 binding molecule of any of the above embodiments, wherein the conjugation moiety is conjugated to said binding molecule, and the conjugation moiety comprises a marker and / or a compound for detection. Preferably, the marker for detection is a radioactive element.

[0039] An embodiment of the present invention provides a kit for the detection of IL-5 protein comprising an IL-5 binding molecule of any of the above embodiments.

[0040] Further, embodiments of the present invention provide the use of an IL-5 binding molecule in the preparation of a medicament that targets IL-5 for the treatment of a disease.

[0041] Preferably, the disease is selected from any of asthma, allergic dermatitis, eczema, arthritis, herpes, chronic idiopathic urticaria, scleroderma, hypertrophic scars, chronic obstructive pulmonary disease, atopic dermatitis, idiopathic pulmonary fibrosis, Kawasaki disease, sickle cell disease, Graves' disease, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and kidney disease. EXAMPLES

[0042] The features and performance of the present invention will be further illustrated by examples below.

[0043] Example 1 Preparation of anti-IL-5 protein single domain antibody A. Construction of an expression vector for human recombinant IL-5 protein A search in NCBI gave the coding sequence of IL-5 with the accession number NM_000879.2. Coding in the coding sequence provided an amino acid sequence with the accession number NP_000870.1. The transmembrane region and extracellular end of the amino acid sequence corresponding to NP_000870.1 were analyzed on the TMHMM and SMART websites, respectively. The analysis results showed that the IL-5 protein is a secreted protein without a transmembrane region, has a signal peptide at positions 1 to 19, and has a total length of 134 amino acids. By gene synthesis, the nucleic acid sequence encoding the amino acids at positions 20 to 134 of the IL-5 protein was cloned into the vector pcDNA3.4. The constructed carrier was Sanger sequenced and aligned with the original sequence. After successful verification, the recombinant plasmid was batch extracted, endotoxin was removed, and transfected into suspended 293F cells for expression and purification of the target protein. The SDS-PAGE analysis result of the purified human recombinant IL-5 protein is shown in Figure 1. In FIG. 1, the markers indicate a standard protein molecular weight gradient, and hrIL-5-cHis indicates human recombinant IL-5 protein having a histidine tag at the carboxy terminus.

[0044] The purity of the expressed and purified human recombinant IL-5 protein is about 90% as can be seen from Figure 1. The IL-5 protein obtained in this example was used for camel immunization and antibody screening.

[0045] B. Construction of anti-IL-5 protein single domain antibody library 600μg of purified human recombinant IL-5 protein obtained in step A was mixed with an equal volume of Freund's complete adjuvant and immunized Inner Mongolia Alxa Bactrian camels once a week for a total of seven times. Except for the first immunization, the remaining six immunizations were performed with 300μg of an equal volume mixture of recombinant IL-5 protein and Freund's incomplete adjuvant to produce anti-IL-5 antibodies in the camels.

[0046] After immunization, 100 mL of peripheral blood lymphocytes were collected from the camel, and RNA was extracted from the lymphocytes. cDNA was synthesized using the extracted total RNA, and heavy chain antibody variable regions (VHH) were amplified by nested PCR using the cDNA as a template. Next, the pMECS vector and the amplified VHH fragments were digested with restriction endonucleases, respectively, and the digested fragments and the vector were ligated. The ligated fragments were transformed into competent cells TG1, and a phage display library of IL-5 protein was constructed. The size of the library (recombinant TG1 cells) was approximately 1 × 10 9 It was determined that.

[0047] C. Screening of anti-IL-5 protein single domain antibodies 200 μL of the recombinant TG1 cells obtained in step B were inoculated into 2×TY medium and cultured. 40 μL of helper phage VCSM13 was added during the culture to infect the TG1 cells, and the cells were cultured overnight to amplify the phages. The next day, the phages were precipitated with PEG / NaCl, and the amplified phages were collected by centrifugation to obtain the amplified phage library.

[0048] 500 μg of IL-5 protein diluted in NaHCO3 (100 mM, pH 8.3) was conjugated to the ELISA plate overnight at 4 °C. Negative control wells were also provided. On the second day, 200 μL of 3% nonfat milk was added for inhibition at room temperature for 2 h. After inhibition, 100 μL of the amplified phage library (approximately 2 × 10 11 The phage particles were added and reacted at room temperature for 1 hour. After 1 hour of reaction, the plate was washed with PBS and 0.05% Tween. (Registered Trademark) The phage was then washed five times with 0.20 to remove unbound phages. The phage that specifically bind to IL-5 protein was dissociated with trypsin at a final concentration of 2.5mg / mL and infected into logarithmic growth phase E. coli TG1 cells. The infected E. coli TG1 cells were cultured at 37°C for 1 hour to produce and recover phages for the next screening. The same screening process was repeated for another round, which could be gradually enriched. The effect of enrichment is shown in Figure 2 and Table 1, as an enrichment efficiency of 10 times or more.

[0049] [Table 1] The results show that the P / N is 2500 and the I / E is 4 for round 3 enrichment.

[0050] D. Screening of specific positive clones for IL-5 by phage ELISA When the enrichment efficiency was 10-fold or more, 400 single colonies were selected from the screened positive clones and inoculated into TB medium containing 100 μg / mL ampicillin in 96-deep-well plates. A blank control was also provided. The plates were grown at 37°C until logarithmic growth phase. Then, IPTG was added at a final concentration of 1 mM and grown overnight at 28°C.

[0051] The antibody crude extract was obtained by osmotic burst. Human recombinant IL-5 protein was diluted in NaHCO3 (100 mM, pH 8.3), and 100 μg of human recombinant IL-5 protein was coated in an ELISA plate at 4°C overnight. 100 μL of the obtained antibody crude extract was transferred to an ELISA plate, to which the antigen (recombinant IL-5 protein) was added and incubated at room temperature for 1 h. The unbound antibody was washed with PBST. 100 μL of mouse anti-HA tag antibody (Thermo Fisher) diluted 1:20000 was added and incubated at room temperature for 1 h. The unbound antibody was washed with PBST. 100 μL of anti-rabbit HRP conjugate (horseradish peroxidase-labeled goat anti-rabbit antibody, Thermo Fisher) diluted 1:20000 was added and incubated at room temperature for 1 h. The unbound antibody was washed with PBST. Horseradish peroxidase color development solution was added and reacted at 37°C for 15 minutes, and a stop solution was added and the absorbance at a wavelength of 450 nm was measured using a microplate reader. When the optical density (OD) of the sample well was more than 5 times higher than that of the control well, the sample well was determined to be a positive clone well. For plasmid extraction and sequencing, the bacteria in the positive clone well were transferred to LB medium containing 100 μg / μL ampicillin on a stirrer. The gene sequence of each clone was analyzed using the sequence alignment software Vector NTI. Strains with the same CDR1, CDR2, and CDR3 sequences were considered to be the same clone, and strains with different sequences were considered to be different clones. Finally, an IL-5 protein-specific single domain antibody was obtained. The single domain antibody has an amino acid sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 that forms the entire VHH. The obtained recombinant plasmid of the single domain antibody (positive plasmid, target sequence) can be expressed in a prokaryotic system to finally obtain a single domain antibody protein.

[0052] E. Purification and expression of IL-5 protein-specific single domain antibodies in host E. coli The positive plasmids (pMECS-VHH) of the different clones obtained by the sequencing analysis in step D were electroporated into E. coli HB2151. They were spread on LB+amp+glucose culture plates (i.e., containing ampicillin and glucose) and cultured overnight at 37°C. A single colony was selected and inoculated into 5 mL of LB medium containing ampicillin and cultured overnight at 37°C on a stirrer. 1 mL of the overnight culture was inoculated into 330 mL of TB medium and cultured at 37°C on a stirrer. Absorbance at a wavelength of 600 nm was measured using a spectrophotometer and recorded as OD600. When the OD600 value was measured in the range of 0.6 to 0.9, 1 M IPTG was added and cultured overnight at 28°C on a stirrer. E. coli was harvested by centrifugation. Crude antibody extracts were obtained by osmotic bursting. The antibodies were purified through a Ni column by affinity chromatography. The purified single domain antibodies are shown in Table 2.

[0053] [Table 2]

[0054] Example 2 Humanization of anti-IL-5 single domain antibodies For humanization of single domain antibodies, humanization was performed by humanization of amino acids on the protein surface and grafting of a universal framework.

[0055] The humanization of amino acids on the protein surface was carried out in the following steps: Using the NbBcII10 antibody (PDB accession number: 3DWT) with homologous sequence as reference, homology modeling was performed for antibody strains 1B3 and 2B3 using Discovery Studio and modeling software. The relative accessibility of amino acids to the solvent was calculated according to the protein three-dimensional structure.

[0056] The grafting of the universal framework for humanization of VHH was performed in the following specific steps. First, a universal framework h-NbBcII10FGLA (PDB accession number: 3EAK) for humanization of VHH was designed and provided based on nanobody NbBcII10 (PDB accession number: 3DWT) according to sequence homology, by humanizing the amino acids on the protein surface with reference to human antibody DP-47, and modifying some amino acids of VHH sequence framework-2 as FGLA. The CDRs of h-NbBcII10FGLA, which is used directly as the framework, were replaced with the CDRs of antibody strains 1B3 and 2B3, respectively, to complete the humanization of the antibody.

[0057] Antibody strains 1B3 and 2B3 were humanized to obtain five humanized antibody strain variants. Tables 3 and 4 show the sequence numbers and amino acid changes of these humanized variants. Amino acid residues were numbered according to the Kabat numbering system. A "○" in Tables 3 and 4 indicates the presence of a mutation (substitution) at that position in the nanobody clone. Specifically, for example, "S11L" means that S (serine) is substituted with L (leucine) at position 11, and "S66T" means that S (serine) is substituted with T (threonine) at position 66. Figure 3 shows the results of the alignment of the humanized sequences.

[0058] [Table 3]

[0059] [Table 4]

[0060] Example 3 F. Construction of eukaryotic expression vector for Fc fusion protein of IL-5 protein single domain antibody The nucleic acid sequence of the single domain antibody screened in step D of Example 1 was obtained by Sanger sequencing. The above base sequence, which was codon-optimized by sequence synthesis, was introduced into the modified vector RJK-V4-hFC.

[0061] RJK-V4-hFC is a universal targeting vector for nanobodies, obtained by fusing the Fc fragment of the heavy chain coding sequence of human IgG (NCBI accession number: AB776838.1) with the commercial vector pcDNA3.4 from Invitrogen (vector information is available at https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcDNA3_4_topo_ta_cloning_kit_man.pdf). The vector contains the hinge CH2 and CH3 regions of the IgG heavy chain. Specific modifications are as follows:

[0062] XbaI and AgeI restriction sites in pcDNA3.4 were selected. A multiple cloning site (MCS) and a 6xHis tag were introduced at the 5' and 3' ends of the Fc fragment coding sequence by overlap PCR, respectively. The above fragment was amplified by PCR using a pair of primers with XbaI and AgeI restriction sites. The amplified fragment with XbaI and AgeI restriction sites from pcDNA3.4 and primers was digested with restriction endonucleases XbaI and AgeI, respectively. The digested vector and the introduced fragment were ligated with T4 ligase, and then the ligated product was transformed into E. coli, amplified, and verified by sequencing to obtain a recombinant eukaryotic expression vector.

[0063] The constructed recombinant eukaryotic expression vector was transformed into DH5α E. coli and cultured with plasmid maxiprep extraction to remove endotoxins. The maxiprep extracted plasmid was identified by sequencing. The confirmed recombinant vector was used for subsequent gene transfer and expression in eukaryotic cells.

[0064] G. Expression of Fc fusion proteins of IL-5 protein-specific single domain antibodies in suspended ExpiCHO-S cells 2.5 × 10 5 ExpiCHO-S™ cells were passaged and expanded at a concentration of 100 / mL. The calculated required volume of cells was transferred to a 500 mL shake flask containing 120 mL (final volume) of ExpiCHO™ Expression Medium. The cells were approximately 4×10 6 ~6×10 6 The day before transfection, ExpiCHO-S™ was cultured at a concentration of 3.5 × 10 viable cells / mL. 6 The cells were diluted to 10 viable cells / mL and cultured overnight. On the day of transfection, the cell density and the percentage of viable cells were measured. Before transfection, the cell density was approximately 7 × 10 6 ~10×10 6 Cells should reach 6 x 10 viable cells / mL. 6 The cells were diluted to 10 ... Approximately 8 days after gene transfection (cell viability was less than 70%), the supernatant was collected.

[0065] H. Expression of anti-IL-5 protein single domain antibody Fc fusion protein in suspended 293F cells Three days before gene transfection, 2.5 × 10 5293F cells were passaged and expanded at a concentration of 1000 / mL. The calculated volume of cells was transferred to a 500 mL shake flask containing 120 mL (final volume) of fresh pre-warmed OPM-293CD05 medium. The cells were approximately 2 × 10 6 ~3×10 6 The cells were cultured to a concentration of 10 ... 6 ~3×10 6 The total number of viable cells / mL should be 1 × 10 6 The cells in the calculated required volume were transferred to a 500 mL shake flask containing 100 mL (final volume) of fresh pre-warmed medium. PEI (1 mg / mL) reagent was diluted in 4 mL of Opti-MEM medium, shaken, and mixed uniformly by pipetting. Plasmid DNA obtained in step F was diluted in 4 mL of Opti-MEM medium, shaken, mixed uniformly, filtered through a 0.22 μm filter head, and incubated at room temperature for 5 min. The diluted PEI reagent was added to the diluted DNA and mixed uniformly by inverting upside down. The PEI / plasmid DNA mixture was incubated at room temperature for 15-20 min and gently added to the prepared cell suspension while gently shaking the shake flask. The cells were shaken and incubated at 120 rpm in 5% CO2 at 37 °C. After 24 and 72 h of transfection, 5 mL of OPM-CHOPFF05 feed was added. Approximately 7 days after gene transfection (cell viability was less than 70%), the supernatant was collected.

[0066] I. Purification of anti-IL-5 protein single domain antibody Fc fusion protein The expression supernatant of the Fc fusion protein obtained in step G or H was filtered through a 0.45 μm disposable filter head to remove insoluble impurities. Using a protein purification device, the filtrate was purified by affinity chromatography with an agarose packing conjugated to protein A based on the binding of the human Fc fusion protein to protein A. The filtrate was passed through a column pre-packed with protein A at a flow rate of 1 mL / min. In this step, the target protein in the filtrate binds to the packing. The impurity proteins bound to the column were washed with low-salt and high-salt buffers. The target protein bound to the column was eluted with a low pH buffer. The eluted solution was quickly added to a Tris-HCl solution at pH 9.0 for neutralization.

[0067] The neutralized protein solution was dialyzed and subjected to SDS-PAGE analysis. Proteins with a purity of 95% or higher and a concentration of 0.5 mg / mL or higher were cryopreserved and ready for use.

[0068] Verification example 1 Determination of the dose-response relationship of the IL-5 protein-specific single domain antibodies provided in Example 1 50 μL of 1 μg / mL IL-5 protein was coated on an ELISA plate overnight at 4°C. The plate was washed. 200 μL of 5% milk was added and incubated at 37°C for 1 hour. The IL-5 protein-specific single domain antibody (VHH) obtained in Example 1 was diluted to 2 μg / mL, and then diluted in a 5-fold gradient to obtain a total of 8 concentration gradients. The plate was washed. 50 μL of the antibody was added to a duplicate plate and incubated at 37°C for 1 hour. The plate was washed. 50 μL of mouse anti-HA tag HRP secondary antibody was added and incubated at 37°C for 30 minutes. The plate was washed multiple times. 50 μL of TMB stored at room temperature was added and reacted at room temperature in the dark for 15 minutes. 50 μL of stop solution (1N HCl) was added. The results were measured and recorded using a microplate reader. The curve was plotted. The concentration of 50% of the maximum effect (EC50), which is the concentration showing 50% of the maximum effect, was calculated, and the results are shown in Figure 4 and Table 5.

[0069] [Table 5]

[0070] Verification example 2 Expression and purification of Tool antibody (Tab) targeting human IL-5 protein Tab1 (control antibody 1) and Tab2 (control antibody 2) were obtained. Tab1 is mepolizumab with the sequence shown in US7982005B2. Tab2 is reslizumab with the sequence shown in US6056957.

[0071] The retrieved Tab1 and Tab2 sequences were submitted to General Biol (Anhui) Co., Ltd. for codon optimization for mammalian cell expression systems and cloned into the pcDNA3.1 vector, respectively.

[0072] Plasmid-positive bacteria screened for resistance were amplified and plasmids were extracted using a plasmid midiprep kit (Macherey Nagel, Cat. No. 740412.50). 100 μg of plasmid (50 μg heavy chain and 50 μg light chain) was added per 100 mL of cells, and the cells were transiently transfected and expressed in 293F cells (FreeStyle 293 Expression Medium, Thermo, Cat. No. 12338026+F-68, Thermo, Cat. No. 24040032) using PEI. 6–24 h after transfection, 5% by volume of 10% Peptron (Sigma, Cat. No. P0521-100G) was added and the cells were cultured at 130 rpm with 8% CO2 for approximately 7–8 days. When cell viability dropped to 50%, the expression supernatant was harvested and purified with a Protein A (GE, Cat. No. 17-5438-02) gravity column. After PBS dialysis, the concentration was measured using Nanodrop, the purity was determined by SEC, and the binding was verified by indirect ELISA. The Tab antibody obtained by this method has a concentration of 2mg / mL or higher and a purity of 94% or higher. The EC50 of binding to IL-5 protein (Novoprotein, Cat. No. CS33) is shown in Figure 5 and Table 6.

[0073] [Table 6]

[0074] Experiments of TF-1 cell proliferation induced by human recombinant IL-5 protein and neutralized by Tool antibody (Tab) Experiments of TF-1 cell proliferation induced by human recombinant IL-5 protein: TF-1 cells, passaged 3-4 times after resuscitation, were seeded in 96-well plates at 10,000 cells / well. Human IL-5 protein was composed of a solution with a maximum concentration of 500ng / mL and diluted in a 5-fold gradient. The gradient-diluted IL-5 protein solution was added to cell culture wells in an equal volume with cell culture medium. After 72 hours of culture, cell viability was detected using a luminescent cell viability detection kit. The EC80 of IL-5-induced TF-1 cell proliferation was calculated according to the detection results. EC80 (concentration of 80% of maximum effect) is the concentration that shows 80% of the maximum effect.

[0075] Experiments on TF-1 cell proliferation induced by Tab-neutralized human IL-5 protein: TF-1 cells, passaged 3-4 times after resuscitation, were seeded in 96-well plates at 10,000 cells / well. Tab1 and Tab2 were composed of 10 μg / mL solutions and diluted in a 5-fold gradient. The gradient-diluted Tab was mixed 1:1 with IL-5 at the EC80 concentration obtained in the proliferation experiment to obtain a mixed solution. The mixed solution was added to cell culture wells in an equal volume to the cell culture medium. After 72 hours of culture, cell viability was detected using a luminescent cell viability detection kit. The EC50 concentrations of TAb1 and Tab2 to neutralize IL-5-induced TF-1 cell proliferation were calculated according to the detection results. The results are shown in Figure 6 and Table 7.

[0076] [Table 7]

[0077] Verification example 3 Detection of IL-5-induced proliferation of TF-1 cells neutralized with Fc fusion protein of anti-IL-5 protein single domain antibody TF-1 cells, which had been passaged 3-4 times after resuscitation, were seeded in 96-well plates at 10,000 cells / well. Tab1, Tab2 and Fc fusion proteins of single domain antibodies provided in Example 3-I were composed of 10 μg / mL solutions and diluted in a 5-fold gradient. The gradient-diluted Tab and single domain antibodies were mixed 1:1 with IL-5 protein at the EC80 concentration obtained in the proliferation experiment to obtain a mixed solution. The mixed solution was added to cell culture wells in an equal volume to the cell culture medium. After 72 hours of culture, cell viability was detected using a luminescent cell viability detection kit. The EC50 concentrations of different single domain antibodies that neutralize IL-5-induced TF-1 cell proliferation were calculated according to the detection results. The results are shown in Figures 7-8 and Tables 8-9.

[0078] [Table 8]

[0079] [Table 9]

[0080] Detection of affinity kinetics of IL-5 binding molecules SD buffer composition: bovine serum albumin and Tween (Registered Trademark) Bovine serum albumin and Tween 20 were added at mass (or volume) fractions of 0.1% and 0.02%, respectively. (Registered Trademark) An appropriate amount of 20 was dissolved in 1×PBS (pH 7.4). The IL-5 binding molecule was constituted at a concentration of 10 μg / mL in SD buffer. Preparation of antigen working solution: antigen was prepared at 200nM in SD buffer, then diluted in a 2-fold gradient, totaling 5 concentration gradients. One blank control of SD buffer was also provided. An appropriate amount of 0.1M glycine stock solution was diluted 10-fold in deionized water and mixed evenly to obtain regeneration solution. Data Acquisiton software in Octet96 and its supporting computer was turned on. The bottom and sides of the uptake probe were washed using lens paper with an appropriate amount of 75% ethanol. The device was pre-heated for more than 15 minutes. Sensor prewetting: The sensor was immersed in SD buffer for at least 10 minutes before testing.Then, the machine procedure was set for the test procedure according to the sequence of baseline → antibody → baseline → antigen binding → antigen dissociation → sensor regeneration.

[0081] The affinity kinetic parameters of the IL-5 binding molecules specifically tested are shown in Table 10.

[0082] [Table 10]

[0083] KD is the affinity constant, with units of molar (M). Ka is the binding rate constant, with units of reciprocal molar time (1 / Ms). Kd is the dissociation rate constant, with units of reciprocal time. R 2 R is the degree of fit, i.e., how well the measured curve and the fitted curve match. 2 The closer to 1, the closer the fitted value is to the actual value. In this system, R 2 shall be at least greater than 0.95. X 2 is a statistical parameter reflecting the value measured by the system. X 2 is less than 3, and X 2 The smaller the value, the more reliable the measurement. Other error values ​​are the error values ​​of the corresponding parameters and are one order of magnitude (10 times) smaller than the corresponding parameters or smaller.

[0084] The results showed that compared to the tool antibody, clone 1B3 had a slightly smaller affinity kinetic constant, the remaining clones had slightly larger affinity kinetic constants, and there were no significant differences in the affinity kinetic constants between the samples tested, with no statistically significant differences.

[0085] What is described in this specification is merely a preferred embodiment of the present invention, and is not intended to limit the present invention. Those skilled in the art can make various changes and modifications to the present invention. Any modifications, equivalent replacements or improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-IL-5 single domain antibody capable of specifically binding to IL-5, comprising a heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3, An anti-IL-5 single domain antibody, the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 being represented by any one of the following (1) to (3): (1) CDR1 having the amino acid sequence represented by SEQ ID NO: 43, CDR2 having the amino acid sequence represented by SEQ ID NO: 52, and CDR3 having the amino acid sequence represented by SEQ ID NO: 59; (2) CDR1 having the amino acid sequence represented by SEQ ID NO: 47, CDR2 having the amino acid sequence represented by SEQ ID NO: 52, and CDR3 having the amino acid sequence represented by SEQ ID NO: 59; and (3) CDR1 having the amino acid sequence shown in SEQ ID NO:43, CDR2 having the amino acid sequence shown in SEQ ID NO:56, and CDR3 having the amino acid sequence shown in SEQ ID NO:

59.

2. The heavy chain variable region further comprises a framework region comprising FR1, FR2, FR3, and FR4; The anti-IL-5 single domain antibody of claim 1, wherein FR1 has the amino acid sequence shown in SEQ ID NO: 65 or 67, FR2 has the amino acid sequence shown in SEQ ID NO: 70 or 72, FR3 has an amino acid sequence selected from the sequences shown in SEQ ID NOs: 73 to 75, 77, 78, 80 and 85, and FR4 has the amino acid sequence shown in SEQ ID NO:

87.

3. The anti-IL-5 single domain antibody according to claim 2, wherein the framework regions FR1, FR2 and FR3 of the heavy chain variable region have any of the sequences shown in the following (14), (17), (23) to (25), (27) and (28): (14) SEQ ID NOs: 65, 72 and 85, (17) SEQ ID NOs: 65, 72 and 73, (23) SEQ ID NOs: 67, 70 and 80, (24) SEQ ID NOs: 65, 72 and 78, (25) SEQ ID NOs: 65, 72 and 77, (27) SEQ ID NOs: 65, 72, and 74, and (28) SEQ ID NOs: 65, 72 and 75.

4. The anti-IL-5 single domain antibody of claim 2, wherein the heavy chain variable region has an amino acid sequence selected from any of the sequences set forth in SEQ ID NOs: 13 to 17 and 19 to 21.

5. The anti-IL-5 single domain antibody of claim 2, wherein the heavy chain variable region is a humanized heavy chain variable region.

6. The anti-IL-5 single domain antibody of claim 1, further comprising an immunoglobulin Fc region linked to the heavy chain variable region.

7. The anti-IL-5 single domain antibody of claim 6, wherein the immunoglobulin Fc region is a human immunoglobulin Fc region.

8. The anti-IL-5 single domain antibody of claim 7, wherein the human immunoglobulin Fc region is a human IgG4 Fc region.

9. An isolated nucleic acid encoding an IL-5 binding molecule according to any one of claims 1 to 8.

10. 10. The isolated nucleic acid of claim 9, wherein the isolated nucleic acid has a sequence selected from any of the sequences set forth in SEQ ID NOs: 34-38 and 40-42.

11. A recombinant vector comprising the isolated nucleic acid of claim 9 or 10.

12. A host cell containing the recombinant vector of claim 11.

13. A method for preparing an anti-IL-5 single domain antibody, comprising culturing the host cell of claim 12 to obtain the anti-IL-5 single domain antibody.

14. A conjugate for binding to an IL-5 protein, comprising a conjugation moiety and an anti-IL-5 single domain antibody according to any one of claims 1 to 8, the conjugation moiety is conjugated to the binding molecule; A conjugate, wherein the conjugation moiety comprises a marker and / or a compound for detection.

15. The conjugate of claim 14, wherein the marker for detection is a radioactive element.

16. A kit for detecting IL-5 protein, comprising an anti-IL-5 single domain antibody according to any one of claims 1 to 8.

17. Use of an anti-IL-5 single domain antibody according to any one of claims 1 to 8 in the preparation of a drug targeting IL-5 for treating a disease.

18. 18. The use according to claim 17, wherein the disease is selected from any of asthma, allergic dermatitis, eczema, arthritis, herpes, chronic idiopathic urticaria, scleroderma, hypertrophic scars, chronic obstructive pulmonary disease, atopic dermatitis, idiopathic pulmonary fibrosis, Kawasaki disease, sickle cell disease, Graves' disease, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, and renal disease.

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