WO2022063281A1 - 结合人il-33的抗体、其制备方法和用途 - Google Patents

结合人il-33的抗体、其制备方法和用途 Download PDF

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WO2022063281A1
WO2022063281A1 PCT/CN2021/120785 CN2021120785W WO2022063281A1 WO 2022063281 A1 WO2022063281 A1 WO 2022063281A1 CN 2021120785 W CN2021120785 W CN 2021120785W WO 2022063281 A1 WO2022063281 A1 WO 2022063281A1
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antibody
seq
human
antigen
binds
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French (fr)
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郭伟
张学赛
徐慧婷
李晴柔
赵乐
陈建鹤
黄浩旻
朱祯平
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三生国健药业（上海）股份有限公司
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Priority to CN202180063091.1A priority Critical patent/CN116234910A/zh
Priority to EP21871650.4A priority patent/EP4219551A1/en
Priority to JP2023518831A priority patent/JP2023542394A/ja
Priority to US18/246,756 priority patent/US20230399394A1/en
Publication of WO2022063281A1 publication Critical patent/WO2022063281A1/zh

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Definitions

  • the invention belongs to the field of biotechnology, and relates to an antibody binding to human IL-33, a preparation method and use thereof.
  • Interleukin 33 is a multifunctional cytokine, a new member of the IL-1 family. Encoded by the IL-33 gene, it is constitutively expressed in structural cells such as smooth muscle cells, epithelial cells, and endothelial cells. In macrophages and dendritic cells, IL-33 can be induced by inflammatory factors. The study found that IL-33 is a bifunctional protein. On the one hand, IL-33 is localized in the nucleus and acts as a transcription factor; on the other hand, IL-33 is secreted outside the cell and acts as a cytokine by interacting with its receptor ST2.
  • IL-33 is a TH-2-type cytokine and is considered to act as an alarmin. By binding to ST2, it induces TH-2 cells to secrete TH-2 types such as IL-4, IL-5 and IL-13. of cytokines.
  • IL-33 can also cause mast cells and basophils to secrete inflammatory cytokines and chemokines, such as IL-1 ⁇ , IL-6, IL-8, TNF ⁇ , etc., resulting in the secretion of TH by NK cells and NKT cells -Type 1 cytokines, such as IFN ⁇ , etc.
  • Asthma is also known as bronchial asthma. Bronchial asthma is a chronic airway inflammation involving a variety of cells and cellular components. Asthma has long been considered to be an airway inflammation driven by CD4+Th-2 cells. However, anti-CD4 antibodies almost completely depleted CD4+ cells but did not completely reduce IL-4, IL-5 or IL-13 production in the lungs of asthmatic mice, suggesting that other cellular sources of these Th-2 cytokines must exist . IL-33 produces IL-5 and IL-13 from ST2-expressing ILC2s, suggesting that IL-5-induced eosinophilia and IL-13 induction can be induced even in the absence of Th-2 cells of mucus production.
  • IL-33 in the lungs of patients with asthma was higher than that of healthy people, and the expression of IL-33 in the lung tissue of patients with severe asthma was particularly obvious.
  • IL-33 promotes collagen synthesis in asthmatic fibroblasts in children with severe asthma, suggesting that IL-33 plays a role in the development of airway remodeling characteristic of severe asthma.
  • Allergic airway inflammation can be alleviated by anti-IL-33 antibody treatment.
  • IL-33 can activate Th-2 cells expressing ST2, IL-5 and IL-13 produced by Th-2 cells and ILC2 cells are involved in the pathogenesis of asthma.
  • the IL-33 pathway has also been implicated in the treatment of various conditions such as atopic/atopic dermatitis, arthritis, chronic sinusitis, chronic obstructive pulmonary disease (COPD), systemic sclerosis, liver fibrosis, psoriasis, Ulcerative colitis, Crohn's disease, multiple sclerosis, diabetic kidney disease, inflammatory bowel disease, psoriasis, eosinophilic esophagitis, diabetic macular edema, age-related macular degeneration, dry eye disease, tumors Wait.
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • systemic sclerosis liver fibrosis
  • psoriasis Ulcerative colitis
  • Crohn's disease multiple sclerosis
  • diabetic kidney disease inflammatory bowel disease
  • psoriasis eosinophilic esophagitis
  • diabetic macular edema age-related
  • the inventors of the present invention have conducted a large number of experiments, from antigen immunization, hybridoma screening, antibody expression and purification to biological activity identification, and obtained the mouse-derived antibody that specifically binds to human IL-33. On the basis, further construct and obtain its chimeric antibody and humanized antibody.
  • the object of the present invention is to provide an antibody that binds to human IL-33 or an antigen-binding fragment thereof; to provide a nucleotide molecule encoding the antibody that binds to human IL-33 or an antigen-binding fragment thereof; to provide a nucleic acid molecule comprising the Expression vector for nucleotide molecule; host cell for providing the expression vector; providing the antibody that binds to human IL-33 or a method for preparing an antigen-binding fragment thereof; providing an antibody that binds to human IL-33 or an antigen-binding fragment thereof A pharmaceutical composition; providing the application of the antibody that binds to human IL-33 or an antigen-binding fragment thereof in preparing a medicine.
  • One aspect of the present invention provides an antibody or antigen-binding fragment thereof that binds human IL-33, wherein the antibody or antigen-binding fragment thereof binds to human IL-33 with an affinity EC50 of less than 1 nM.
  • the light chain of the antibody has the L-CDR2 shown in SEQ ID No: 22, and has the following characteristics:
  • the Kd value of the antibody for human IL33 is much smaller than the Kd value for mouse IL33 (a difference of 20 times or more).
  • the antibody includes:
  • the amino acid sequence of said L-CDR1 is as shown in SEQ ID NO: 21 or a mutation of SEQ ID NO: 21 up to 2 amino acid substitution mutations
  • the amino acid sequence of the L-CDR2 is shown in SEQ ID NO: 22
  • the amino acid sequence of the L-CDR3 is shown in SEQ ID NO: 23 or the mutant of SEQ ID NO: 23 with up to 3 amino acid substitution mutations .
  • the antibody or antigen-binding fragment thereof that binds to human IL-33 includes:
  • H-CDR1, H-CDR2, H-CDR3 the amino acid sequences of the H-CDR1, H-CDR2, H-CDR3 are shown in SEQ ID NOs: 15, 16 and 17, respectively, either as set forth in SEQ ID NOs: 18, 19 and 20, respectively, or as set forth in SEQ ID NOs: 18, 24 and 25, respectively, and
  • the antibody or antigen-binding fragment thereof that binds to human IL-33 includes:
  • H-CDR1, H-CDR2, H-CDR3 heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of said H-CDR1, H-CDR2, H-CDR3 are shown in SEQ ID NOs: 18, 24 and 25, respectively, and
  • the antibody or antigen-binding fragment thereof that binds to human IL-33 includes:
  • H-CDR1, H-CDR2, H-CDR3 heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of said H-CDR1, H-CDR2, H-CDR3 are shown in SEQ ID NOs: 18, 24 and 25, respectively, and
  • the amino acid sequence of any of the above CDRs comprises a derived CDR sequence that has been added, deleted, modified and/or substituted by 1, 2, 3, 4, 5, 6 or 7 amino acids, and the amino acid sequence containing all
  • the derivatized antibody composed of the VH and VL of the derived CDR sequences can retain the affinity for binding to IL-33.
  • an “antibody (Ab)” of the invention is a heterotetraglycan protein of about 150,000 Daltons consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. At one end of each heavy chain is a variable region (VH) followed by a constant region.
  • VH variable region
  • Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is opposite the first constant domain of the heavy chain, and the variable domain of the light chain is opposite the variable domain of the heavy chain .
  • Antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) formed from at least two types of antibodies, and the like.
  • a “monoclonal antibody” of the present invention refers to an antibody obtained from a substantially homogeneous population, ie, the individual antibodies contained in the population are identical except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific to a single antigenic site. Also, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they are synthesized by hybridoma cultures without contamination by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characteristically obtained from a substantially homogeneous population of antibodies, which should not be construed as requiring any particular method to produce the antibody.
  • the "antigen-binding fragment” of the present invention refers to a fragment of an antibody that can specifically bind to human IL-33.
  • antigen-binding fragments of the present invention include Fab fragments, F(ab') 2 fragments, Fv fragments, and the like.
  • Fab fragments are fragments produced by papain digestion of antibodies.
  • F(ab') 2 fragments are fragments produced by pepsin digestion of antibodies.
  • Fv fragments are composed of dimers of the heavy and light chain variable regions of an antibody in close non-covalent association.
  • the antibody is a murine antibody, a chimeric antibody or a humanized antibody.
  • the "murine antibody” of the present invention refers to an antibody derived from a rat or a mouse, preferably a mouse.
  • the murine antibody of the present invention is obtained by using human IL-33 as an antigen to immunize mice and screen hybridoma cells.
  • a “chimeric antibody” of the present invention refers to an antibody comprising heavy and light chain variable region sequences derived from one species and constant region sequences derived from another species, eg, having murine heavy and light chains linked to human constant regions Chain variable region antibodies.
  • the chimeric antibody of the present invention is composed of murine antibody 864F3, 874F7, 871G1 heavy chain variable region and then human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region containing mutation recombination, light chain variable region with human IgG1, IgG2, IgG3 or IgG4 The kappa chain constant region was obtained by recombination.
  • Humanized antibody of the present invention means that its CDRs are derived from a non-human species (preferably mouse) antibody, and the remaining parts of the antibody molecule (including framework regions and constant regions) are derived from human antibodies. In addition, framework region residues can be altered to maintain binding affinity.
  • the humanized antibody of the present invention is recombined from the CDR regions of murine antibodies 864F3, 874F7 and 871G1 and the non-CDR regions derived from human antibodies, and the heavy chain variable region is then combined with human IgG1, IgG2, IgG3 or The IgG4 heavy chain constant region is recombined, the light chain variable region is recombined with the human kappa chain constant region, and some important residues are obtained by mutation.
  • the antigen-binding fragments include Fab fragments, F(ab') 2 fragments, and Fv fragments.
  • amino acid sequences of the variable region of the heavy chain and the variable region of the light chain of the antibody that binds to human IL-33 or its antigen-binding fragment are shown in SEQ ID NO: 2 and SEQ ID NO: 6, respectively. , or as shown in SEQ ID NO: 4 and SEQ ID NO: 6, respectively, or as SEQ ID NO: 8 and SEQ ID NO: 10, respectively, or as SEQ ID NO: 4 and SEQ ID NO: 12, respectively Show.
  • amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment that binds to human IL-33 is shown in SEQ ID NO: 32, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: : 29, 30, 31, 33, 34 or 35.
  • amino acid sequence of the light chain variable region of the antibody that binds to human IL-33 or its antigen-binding fragment is shown in SEQ ID NO: 40
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: : shown at 37, 38 or 39.
  • the heavy chain constant region of the antibody is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.
  • amino acid sequence of the light chain variable region of the antibody that binds to human IL-33 or its antigen-binding fragment is shown in SEQ ID NO: 36
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. : shown at 33, 34 or 35.
  • the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment that binds to human IL-33 is shown in SEQ ID NO: 32, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 32. ID NO: 34; or the amino acid sequence of the light chain variable region of the antibody that binds to human IL-33 or its antigen-binding fragment is shown in SEQ ID NO: 40, and the amino acid sequence of the heavy chain variable region is as follows SEQ ID NO:38.
  • the amino acid sequence of the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • the amino acid sequence of the heavy chain variable region is at least the same as the amino acid sequence shown in SEQ ID NO: 29, 30, 31, 33, 34, 35, 37, 38 or 39 in the sequence listing. 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • amino acid sequences of the heavy chain constant region and the light chain constant region of the antibody or antigen-binding fragment thereof that bind to human IL-33 are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.
  • the binding epitope of the antibody or antigen-binding fragment thereof that binds to human IL-33 and IL-33 protein comprises a site corresponding to SEQ ID NO.55 selected from the group consisting of:
  • Lysine 45 (K45), Valine 49 (V49), Aspartic acid 65 (D65), Leucine 50 (L50), Serine 60 (S60), Serine 52 (S52), Lysine 48 (K48), Leucine 51 (L51), Tyrosine 53 (Y53), Glutamic acid 55 (E55);
  • lysine at position 45 K45
  • valine at position 49 V49
  • aspartic acid at position 65 D65
  • leucine at position 50 L50
  • the site corresponds to the amino acid sequence of wild-type human IL-33 protein, and the amino acid sequence is Ser 112 to Thr 270 of NCBI:NP_254274.1.
  • Another aspect of the present invention provides a nucleotide molecule encoding the above-mentioned human IL-33-binding antibody or an antigen-binding fragment thereof.
  • nucleotide sequences of the nucleotide molecule encoding the variable region of the heavy chain and the variable region of the light chain are shown in SEQ ID NO: 1 and SEQ ID NO: 5, respectively, or as shown in SEQ ID NO: 5, respectively.
  • nucleotide sequence of the nucleotide molecule encoding the variable region of the heavy chain is shown in SEQ ID NO: 41, 42, 43, 45, 46 or 47, respectively, and the nucleotide sequence encoding the variable region of the light chain is shown in The nucleotide sequence is shown in SEQ ID NO:44.
  • nucleotide sequence encoding the variable region of the heavy chain of the nucleotide molecule is shown in SEQ ID NO: 49, 50 or 51, respectively, and the nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO: 49, 50 or 51, respectively. NO: 52 shown.
  • nucleotide sequence of the nucleotide molecule encoding the variable region of the heavy chain is shown in SEQ ID NO: 45, 46 or 47 respectively
  • nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO: 45, 46 or 47 respectively.
  • the preparation method of the nucleotide molecule of the present invention is a conventional preparation method in the field, and preferably includes the following preparation method: obtaining a nucleotide molecule encoding the above-mentioned monoclonal antibody by gene cloning technology such as PCR method, or by The nucleotide molecule encoding the above-mentioned monoclonal antibody is obtained by the method of artificial whole sequence synthesis.
  • nucleotide sequence encoding the amino acid sequence of the above-mentioned human IL-33-binding antibody or antigen-binding fragment thereof can be appropriately introduced into the nucleotide sequence encoding the amino acid sequence of the above-mentioned human IL-33-binding antibody or antigen-binding fragment thereof to provide a polynucleotide homology thing.
  • homologs of the polynucleotides of the present invention can be prepared by substituting, deleting or adding one or more bases of the gene encoding the human IL-33-binding antibody or antigen-binding fragment thereof within a range that maintains the activity of the antibody have to.
  • Another aspect of the present invention provides an expression vector containing the above-mentioned nucleotide molecule.
  • the expression vector is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate control sequences. sequence of expression vectors.
  • the expression vector may be a virus or plasmid, such as a suitable phage or phagemid, for more technical details see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989. See Current Protocols in Molecular Biology, 2nd ed., Ausubel et al., for many known techniques and protocols for nucleic acid manipulation.
  • the expression vector of the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR, pcDNA4, pDHFF, pGM-CSF or pCHO 1.0.
  • the present invention further provides a host cell containing the above-mentioned expression vector.
  • the host cells described in the present invention are various conventional host cells in the field, as long as the above-mentioned recombinant expression vector can stably replicate by itself, and the nucleotides it carries can be effectively expressed.
  • the host cells include prokaryotic expression cells and eukaryotic expression cells, and the host cells preferably include: COS, CHO (Chinese Hamster Ovary, Chinese Hamster Ovary), NSO, sf9, sf21, DH5 ⁇ , BL21 (DE3) Or TG1, more preferably E. coli TG1, BL21(DE3) cells (expressing single chain antibody or Fab antibody) or CHO-K1 cells (expressing full-length IgG antibody).
  • the preferred recombinant expression transformants of the present invention can be obtained by transforming the aforementioned expression vector into a host cell.
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a thermal shock method or an electrotransformation method.
  • Another aspect of the present invention provides the above-mentioned method for binding human IL-33 antibody or its antigen-binding fragment, characterized in that, the method comprises the following steps:
  • the method for culturing host cells and the method for separating and purifying the antibody of the present invention are conventional methods in the art. For specific operation methods, please refer to the corresponding cell culture technical manual and antibody separation and purification technical manual.
  • the preparation method of the antibody that binds to human IL-33 or its antigen-binding fragment disclosed in the present invention comprises: under expression conditions, culturing the above-mentioned host cell, so as to express the antibody that binds to human IL-33 or its antigen-binding fragment ; Isolation and purification of the described antibody or antigen-binding fragment thereof that binds to human IL-33.
  • the recombinant protein can be purified to a substantially homogeneous material, eg, as a single band on SDS-PAGE.
  • the antibodies that bind to human IL-33 or their antigen-binding fragments thereof disclosed in the present invention can be separated and purified by affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high-salt buffer can be used.
  • the human IL-33-binding antibody or its antigen-binding fragment bound on the affinity column is eluted by methods such as liquid solution, pH change and the like.
  • the inventors of the present invention carried out a detection experiment on the obtained antibody that binds to human IL-33 or its antigen-binding fragment, and the experimental results show that the said antibody that binds to human IL-33 or its antigen-binding fragment can effectively Binds to antigen with high affinity.
  • compositions comprising the above-mentioned human IL-33-binding antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.
  • the antibody or antigen-binding fragment thereof that binds to human IL-33 provided by the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert a more stable curative effect.
  • 33 the conformational integrity of the antibody or antigen-binding fragment thereof, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation).
  • degradation including but not limited to aggregation, deamination, or oxidation.
  • it is usually stable at 2°C-8°C for at least one year, and for lyophilized formulations, it is stable at 30°C for at least six months.
  • the bispecific antibody preparations can be suspension, water injection, freeze-dried and other preparations commonly used in the pharmaceutical field.
  • pharmaceutically acceptable carriers preferably include, but are not limited to, surfactants, solution stabilizers, and isotonicity regulators and one or a combination of buffers.
  • the surfactant preferably includes but is not limited to: non-ionic surfactant such as polyoxyethylene sorbitan fatty acid ester (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium lauryl sulfate (SDS); sodium lauryl sulfate; myristyl, linoleyl, or octadecyl sarcosine; Pluronics; The trend is minimal.
  • non-ionic surfactant such as polyoxyethylene sorbitan fatty acid ester (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium lauryl sulfate (SDS); sodium lauryl sulfate; myristyl, linoleyl, or octadecyl sarcosine; Pluronics; The trend is minimal.
  • non-ionic surfactant such as polyoxyethylene sorbitan
  • Solution stabilizers preferably include, but are not limited to, one or a combination of the following: sugars, such as reducing sugars and non-reducing sugars; amino acids, such as monosodium glutamate or histidine; alcohols, For example: trihydric alcohol, higher sugar alcohol, propylene glycol, polyethylene glycol, etc., the added amount of the solution stabilizer should make the final formed preparation maintain a stable state within the time that those skilled in the art think it is stable.
  • the isotonicity modifier preferably includes, but is not limited to, one of sodium chloride, mannitol, or a combination thereof.
  • the buffer preferably includes, but is not limited to, one of Tris, histidine buffer, phosphate buffer, or a combination thereof.
  • Another aspect of the present invention provides an antibody-drug conjugate, the antibody-drug conjugate comprising:
  • a conjugation moiety conjugated to the antibody moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • Another aspect of the present invention provides the above-mentioned antibody that binds to human IL-33 or its antigen-binding fragment or pharmaceutical composition, and the antibody-drug conjugate in preparation for the treatment of asthma, arthritis, atopic/allergic dermatitis, chronic sinusitis , chronic obstructive pulmonary disease (COPD), systemic sclerosis, liver fibrosis, psoriasis, ulcerative colitis, Crohn's disease, multiple sclerosis, diabetic kidney disease, inflammatory bowel disease, psoriasis, Drug application in eosinophilic esophagitis, diabetic macular edema, age-related macular degeneration, dry eye disease, and tumors.
  • the arthritis includes rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gouty arthritis, reactive arthritis, infectious arthritis, traumatic arthritis, psoriatic arthritis, enteropathic arthritis .
  • the dose to be administered depends on the age and weight of the patient, the nature and severity of the disease, and the route of administration However, you can refer to the results of animal experiments and various situations, and the total dose cannot exceed a certain range. Specifically, the dose for intravenous injection is 1-1800 mg/day.
  • Another aspect of the present invention provides a treatment for asthma, arthritis, atopic/allergic dermatitis, chronic sinusitis, chronic obstructive pulmonary disease (COPD), systemic sclerosis, liver fibrosis, psoriasis, ulcerative colitis Methods for , Crohn's disease, multiple sclerosis, diabetic kidney disease, inflammatory bowel disease, psoriasis, eosinophilic esophagitis, diabetic macular edema, age-related macular degeneration, dry eye disease, tumors, which It is characterized in that the above-mentioned antibody that binds to human IL-33 or an antigen-binding fragment thereof or a pharmaceutical composition, an antibody-drug conjugate, or a combination thereof is administered to a subject in need.
  • COPD chronic obstructive pulmonary disease
  • Another aspect of the present invention provides a mutant of IL-33 protein, corresponding to the amino acid sequence of wild-type human IL-33 protein, wherein the mutant comprises mutation at one or more sites selected from the group consisting of;
  • sequence of the wild-type human IL-33 protein is shown in SEQ ID NO.55.
  • the mutant comprises a mutation at one or more sites selected from the group consisting of;
  • the mutant comprises a mutation at one or more sites selected from the group consisting of;
  • the mutant comprises a mutation at one or more sites selected from the group consisting of;
  • the affinity of the mutant IL-33 protein to the antibody that binds to human IL-33 is reduced by 1 times compared with the affinity of the wild-type IL-33 protein to the antibody that binds to human IL-33, Preferably 5 times, more preferably 10 times, more preferably 25 times, and optimally 50 times.
  • the mutant further comprises a mutation at one or more sites selected from the following group;
  • the mutant of the IL-33 protein comprises mutating one or more amino acids in (Z1)-(Z27) to alanine (A) or glycine (G).
  • the mutation in the IL-33 protein mutant is selected from the following group:
  • the mutant of the IL-33 protein is selected from the following group:
  • the amino acid sequence of the IL-33 protein mutant is at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% compared with SEQ ID NO.55 %, 96%, 97%, 98%, 99% or more sequence identity.
  • Another aspect of the present invention provides a method for evaluating the binding epitope of an anti-human IL-33 antibody, comprising:
  • Heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of said H-CDR1, H-CDR2, H-CDR3 are shown in SEQ ID NOs: 18, 24 and 25, respectively, and
  • Light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequences of said L-CDR1, L-CDR2, L-CDR3 are as SEQ ID NOs: 26, 22 and 27, respectively.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the positive improvement effect of the present invention is: at present, it is urgent to develop new, specific and efficient therapeutic drugs for diseases with strong expression of IL-33, so as to improve the quality of life of people suffering from such diseases, and provide more and more effective drugs for patients.
  • the antibody of the present invention has good affinity for human IL-33, can block the binding of IL-33 and its receptor ST2, can be used for the treatment of various diseases, and has a good clinical application prospect.
  • Figures 1A-B Binding activity of murine antibodies to human IL-33.
  • Figures 2A-B Murine antibodies block ST2 binding activity to IL-33.
  • Figures 3A-C Inhibitory effects of murine antibodies on IL-33-induced IL-6 expression in HUVEC cells.
  • Figure 4 Inhibitory effect of murine antibody on IL-33-induced IL-8 expression in HUVEC cells.
  • Figure 5 Cross-activity of murine antibodies with cynomolgus monkey IL-33 antigen.
  • Figure 6 Spleen weighing assay to evaluate in vivo neutralizing activity of murine antibodies.
  • Figure 7 Binding activity of chimeric antibodies to human IL-33.
  • Figure 8 Chimeric antibodies block ST2 binding activity to IL-33.
  • Figures 9A-D Binding activity of each humanized antibody to human IL-33.
  • Figure 10 Humanized antibodies block ST2 binding activity to IL-33.
  • Figure 11 Inhibitory effect of humanized antibodies on IL-33-induced IL-6 expression in HUVEC cells.
  • Figure 12 Inhibitory effect of humanized antibodies on IL-33-induced IFN ⁇ secretion by PBMC.
  • Figure 13 Inhibitory effect of humanized antibodies on IL-33-induced IFN ⁇ secretion by NK cells.
  • Figure 14 Humanized antibodies 864F3-Hu4 and 874F7-Hu1 can effectively inhibit IL-33-induced IL-5 secretion by KU812 cells.
  • Figure 15 Humanized antibodies 864F3-Hu4 and 874F7-Hu1 can effectively inhibit IL-33-induced IL-13 secretion by KU812 cells.
  • Figure 16 Spleen weighing assay to evaluate in vivo pharmacodynamic activity of humanized antibodies.
  • Figure 17 Spleen weighing assay to evaluate the in vivo pharmacodynamic activity of humanized antibodies 864F3-Hu4 and 874F7-Hu1.
  • Figure 18 Humanized antibodies 864F3-Hu4 and 874F7-Hu1 can effectively inhibit mouse peripheral blood IL-5 secretion.
  • Figure 19 Humanized antibodies 864F3-Hu4 and 874F7-Hu1 can effectively reduce the increase of eosinophils in mouse peripheral blood induced by stimulation with human IL-33.
  • Figure 20 Affinity of IL-33 mutant proteins (E44A, K45A, K46A, Q57A, H58A, P59A, S60A) for 874F7-Hul.
  • Figure 21 Affinity of IL-33 mutant proteins (K48A, V49A, L51A, S52A, Y53A, E55A) for 874F7-Hul.
  • Figure 22 Affinity of IL-33 mutant proteins (N61A, E62A, S63A, D65A, V67A, D68A, K70A) for 874F7-Hul.
  • Figure 23 Affinity of IL-33 mutant protein (L50A) for 874F7-Hul.
  • Figure 24 Positions of key amino acid sites affecting 874F7-Hu1 binding in the 3D structure of IL-33 crystallography.
  • the present inventors obtained a series of anti-IL-33 humanized antibodies with excellent affinity.
  • the humanized antibody of the present invention can block the binding of IL-33 and its receptor ST2, inhibit IL-33-induced PBMC and NK cells to secrete IFN ⁇ , and can inhibit IL-33-induced secretion of basophilic leukemia cells KU812 IL-5 and IL-13.
  • the humanized antibodies of the present invention have significant neutralizing activity in vivo. In the mouse animal experiment, the humanized antibody of the present invention can effectively reduce the increase of eosinophils in the peripheral blood of mice caused by stimulation of human IL-33 in a single treatment.
  • the antibodies of the present invention are expected to be useful in the treatment of various IL-33-related disorders. The present invention has been completed on this basis.
  • the terms "antibody (abbreviation Ab)” and “immunoglobulin G (abbreviation IgG)” are heterotetraglycan proteins with the same structural characteristics, which are composed of two identical light chains (L ) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. At one end of each heavy chain is a variable region (VH) followed by a constant region, which consists of three domains, CH1, CH2, and CH3.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end.
  • the constant region of the light chain includes a domain CL; the constant region of the light chain is paired with the CH1 domain of the constant region of the heavy chain, and the light chain can
  • the variable regions are paired with the variable regions of the heavy chain.
  • the constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC), etc.
  • Heavy chain constant regions include IgGl, IgG2, IgG3, IgG4 subtypes; light chain constant regions include kappa (Kappa) or lambda (Lambda).
  • the heavy and light chains of the antibody are covalently linked together by a disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are interpolypeptide disulfide formed between the hinge regions. The bonds are covalently linked together.
  • the terms "Fab” and “Fc” mean that papain can cleave an antibody into two identical Fab segments and one Fc segment.
  • the Fab fragment consists of the VH and CH1 of the heavy chain and the VL and CL domains of the light chain of the antibody.
  • the Fc segment can be called a fragment crystallizable (Fc), which consists of the CH2 and CH3 domains of the antibody.
  • the Fc segment has no antigen-binding activity and is the site where antibodies interact with effector molecules or cells.
  • scFv is a single chain antibody (single chain antibody fragment, scFv).
  • variable means that certain portions of the variable regions of antibodies differ in sequence, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the variable regions of the heavy and light chains. The more conserved part of the variable region is called the frame region (FR).
  • CDRs complementarity-determining regions
  • FR frame region
  • the variable regions of native heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form linking loops, and in some cases can form part of a ⁇ -sheet structure.
  • the CDRs in each chain are tightly packed together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • FR framework region
  • the light and heavy chains of immunoglobulins each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively.
  • a light chain variable domain may thus be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain can thus be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H).
  • the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is substantially identical to a naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96% , 97%, 98% or 99%.
  • human framework region is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to that of a naturally occurring human antibody .
  • linker refers to insertion into an immunoglobulin domain to provide sufficient mobility for the domains of the light and heavy chains to fold into an exchange of one or more amino acid residues of a dual variable region immunoglobulin base.
  • preferred linkers refer to linkers Linker1 and Linker2, wherein Linker1 connects the VH and VL of a single chain antibody (scFv), and Linker2 is used to connect the scFv to the heavy chain of another antibody.
  • linkers examples include monoglycine (Gly), or serine (Ser) residues, and the identity and sequence of amino acid residues in the linker can vary depending on the type of secondary structural elements that need to be implemented in the linker.
  • the antibody of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the bispecific antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, most preferably Preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.
  • conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.
  • the terms “anti”, “binding” and “specific binding” refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it is directed against.
  • the antibody binds the antigen with an equilibrium dissociation constant (KD) of less than about 10-7 M, eg, less than about 10-8 M, 10-9 M, 10-10 M, 10-11 M, or less.
  • KD equilibrium dissociation constant
  • the term “KD” refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.
  • surface plasmon resonance Surface Plasmon Resonance, abbreviated SPR
  • SPR Surface Plasmon Resonance
  • epitope refers to a polypeptide determinant that specifically binds to an antibody.
  • An epitope of the present invention is a region of an antigen that is bound by an antibody.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavitary injection.
  • pharmaceutical composition means that the bispecific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert a more stable curative effect, and these preparations can ensure the bispecific disclosed in the present invention.
  • the conformational integrity of the amino acid core sequence of an antibody while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation).
  • the pharmaceutical composition of the present invention contains a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) of the above-mentioned bispecific antibody (or its conjugate) of the present invention and A pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the bispecific antibodies of the invention can also be used with other therapeutic agents.
  • a safe and effective amount of the bispecific antibody or immunoconjugate thereof is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • ADCs Antibody-Drug Conjugates
  • the present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.
  • ADC antibody-drug conjugate
  • the antibody-drug conjugate includes the antibody, and an effector molecule, and the antibody is conjugated to the effector molecule, preferably chemically conjugated.
  • the effector molecule is preferably a drug with therapeutic activity.
  • the effector molecule can be one or more of a toxin, a chemotherapeutic drug, a small molecule drug, or a radionuclide.
  • the antibody of the present invention and the effector molecule can be coupled through a coupling agent.
  • the coupling agent may be any one or more of non-selective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds.
  • the non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde and the like.
  • the coupling agent utilizing the carboxyl group may be any one or more of a cis-aconitic anhydride type coupling agent (such as cis-aconitic anhydride) and an acyl hydrazone type coupling agent (the coupling site is an acyl hydrazone).
  • antibodies are used to link with various functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes) , stabilizers (eg, ethylene glycol polymers) and therapeutic agents.
  • imaging reagents such as chromophores and fluorophores
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers eg, ethylene glycol polymers
  • therapeutic agents eg, ethylene glycol polymers
  • Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg, drugs, detection reagents, stabilizers
  • the functional agent can be attached to the antibody either directly or indirectly through a linker.
  • Antibodies can be conjugated to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC contains a linker between the drug and the antibody.
  • Linkers can be degradable or non-degradable linkers. Degradable linkers are typically susceptible to degradation in the intracellular environment, eg, at the target site, where the linker is degraded, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (eg, lysosomal or endosomal proteases), or sugar linkers that, for example, can be degraded by glucuronides Enzymatically degraded glucuronide-containing linkers.
  • Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine.
  • degradable linkers include, for example, pH sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, eg, hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers).
  • Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.
  • the linker Before being attached to the antibody, the linker has a reactive reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive reactive group.
  • Sulfhydryl-specific reactive groups are preferred and include, for example, maleimides, haloamides (eg, iodo, bromo, or chloro); haloesters (eg, iodo, bromo, or chloro) ); halogenated methyl ketones (eg iodo, bromo or chloro), benzyl halides (eg iodo, bromo or chloro); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6- bis-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • Linkers can include, for example, maleimide
  • the drug can be any cytotoxic, cytostatic or immunosuppressive drug.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker.
  • the drug is directly attached to the linker, the drug has a reactive reactive group prior to attachment to the antibody.
  • Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors , Vinca alkaloids, etc.
  • drug-linkers can be used to form ADCs in one simple step.
  • bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, a cysteine residue reacts with the reactive moiety of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form the ADC.
  • functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety.
  • azide-based moieties can be used to specifically react with reactive alkynyl groups on drug moieties.
  • the drug is covalently bound to the linker through a 1,3-dipolar cycloaddition between the azide and the alkynyl group.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines).
  • the present invention also provides a method for preparing an ADC, which may further include: combining the antibody with the drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the invention comprise binding the antibody to a bifunctional linker compound under conditions sufficient to form the antibody-linker conjugate. In these embodiments, the methods of the invention further comprise: conjugating the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.
  • the antibody drug conjugate ADC is represented by the following molecular formula:
  • Ab is an antibody
  • D is a drug
  • Balb/c mice purchased from Shanghai Lingchang Biotechnology Co., Ltd.
  • PBMC purchased from Ocellus Biotechnology Shanghai Co., Ltd., item number PB004-C.
  • Human IL-33-his Ser112-Thr270 of the IL-33 sequence (NCBI accession number NP_254274.1) was cloned into the E. coli expression vector for expression, and a 6 ⁇ His tag was added to the C-terminus of IL-33 for It was purified by Ni + affinity chromatography column, and its amino acid sequence is shown in SEQ ID NO:53.
  • Cynomolgus monkey IL-33-his purchased from Yiqiao Shenzhou, item number 90912-CNAE.
  • IL-33-his-biotin protein purchased from Thermo Fisher, Cat. No. 20217, biotinylated IL-33-his protein according to the instructions of EZ-Link NHS-Biotin Reagent reagent.
  • Rat-Anti-human IL-6 Available from BD Pharmingen, Cat. No. 554543.
  • Mouse-Anti-human IFN ⁇ purchased from BD Biosciences, Cat. No. 551221.
  • biotin mouse-anti-human IFN ⁇ purchased from BD Biosciences, Cat. No. 554550.
  • Goat anti-mouse IgG secondary antibody purchased from Millipore, Cat. No. AP181P.
  • HRP-anti human IgG Fc secondary antibody purchased from Sigma, Cat. No. A0170.
  • HRP-labeled streptavidin secondary antibody purchased from BD Biosciences, Cat. No. 554066.
  • IL-6 Kit Invitrogen, Cat. No. 88-7066-77.
  • IL-12 purchased from Sino Biological, Cat. No. CT011-H08H.
  • ST2-Fc protein The human ST2 sequence (NCBI accession number is NP_003847.2) and the Fc region sequence of human IgG1 were fused and cloned into the eukaryotic expression vector PTT5, which was fused and expressed by transfecting HEK-293F cells, and then collected The expression supernatant was purified by ProteinA affinity chromatography.
  • the ST2-Fc amino acid sequence is shown in SEQ ID NO:54.
  • TMB purchased from BD Company, item number 555214.
  • Glutmine glutamine
  • Sodium pyruvate sodium pyruvate
  • MEM-NEAA minimal minimal medium-non-essential amino acid solution
  • Penicillin-streptomycin penicillin-streptomycin
  • FBS fetal bovine serum
  • SA protein Streptavidin, purchased from Sigma, Cat. No. 85878-1MG.
  • SFM medium purchased from Life Technologies, Cat. No. 12045-076.
  • RPMI1640 complete medium purchased from Gibco.
  • Electrofusion apparatus purchased from BTX Company.
  • Microplate reader purchased from Molecular Devices, model SpectraMax 190.
  • the antibody sequence of the present invention is shown in the following table:
  • Balb/c mice were routinely immunized intraperitoneally with prokaryotic recombinantly expressed human IL-33-his protein.
  • soluble human IL-33-his protein and Freund's complete adjuvant emulsification or water-soluble adjuvant (quick antibody) were thoroughly mixed, and then Balb/c mice were injected intraperitoneally (human IL-33-his 50 ⁇ g / mouse), on the fourteenth day, after the soluble human IL-33-his protein was emulsified with incomplete Freund's adjuvant or the water-soluble adjuvant (quick antibody) was thoroughly mixed, the Balb/c mice were boosted by intraperitoneal immunization ( Human IL-33-his 50 ⁇ g/mouse), on the thirty-sixth day, the animals were boosted with soluble human IL-33-his protein as before (human IL-33-his 50 ⁇ g/mouse), three weeks later intraperitoneally The mice were injected with human IL
  • mice Three to four days after the last shock immunization of the mice, the mouse spleen cells and the mouse myeloma cells SP2/0 were electrofused by an electrofusion apparatus using a conventional hybridoma technology scheme. The fused cells are evenly suspended in complete medium.
  • the complete medium is to mix RPMI1640 and DMEM F12 medium 1:1, then add 1% Glutmine (glutamine), 1% Sodium pyruvate (sodium pyruvate), 1 %MEM-NEAA (minimal minimal medium-non-essential amino acid solution), 1% Penicillin-streptomycin (penicillin-streptomycin), 50 ⁇ M ⁇ -mercaptoethanol and 20% FBS (fetal bovine serum) medium; press 10 5 cells/100 ⁇ l/well were divided into 36 96-well culture plates and cultured overnight. The next day, 100 ⁇ l of complete medium containing 2 ⁇ HAT was added to each well, so that the medium in the 96-well plate was 200 ⁇ l/well. (with 1 ⁇ HAT). After 7-12 days, the supernatant was harvested, and hybridoma wells positive for human IL-33-his binding activity were screened by indirect enzyme-linked immunosorbent assay (ELISA).
  • ELISA indirect enzyme-linked immunosorbent
  • the indirect enzyme-linked immunosorbent assay method for screening hybridoma wells with positive human IL-33-his binding activity is as follows: the recombinant human IL-33-his protein is coated with a coating solution (50 mM carbonate coating buffer solution) , pH 9.6) diluted to 1 ⁇ g/ml, 100 ⁇ l/well was added to the ELISA plate, and coated overnight at 4°C. The plate was washed three times with PBST, 200 ⁇ l/well of blocking solution (2% BSA-PBS) was added, and the plate was placed at 37° C. for 1 h and washed once with PBST for use.
  • a coating solution 50 mM carbonate coating buffer solution
  • pH 9.6 pH 9.6
  • 100 ⁇ l/well 100 ⁇ l/well was added to the ELISA plate, and coated overnight at 4°C.
  • the plate was washed three times with PBST, 200 ⁇ l/well of blocking solution (2% BSA-PBS) was added, and
  • the collected hybridoma supernatants were sequentially added to the blocked ELISA plate, 100 ⁇ l/well, and placed at 37° C. for 1 h. Wash the plate 3 times with PBST, add HRP-labeled goat anti-mouse IgG secondary antibody, and place at 37°C for 30 min; after washing the plate 5 times with PBST, pat the remaining droplets dry on absorbent paper as much as possible, and add 100 ⁇ l of TMB to each well, at room temperature ( 20 ⁇ 5°C) in the dark for 5 min; add 50 ⁇ l of 2M H 2 SO 4 stop solution to each well to stop the substrate reaction, read the OD value at 450 nm of the microplate reader, and analyze the binding of the antibody to be tested to the target antigen human IL-33-his ability.
  • the 10 hybridoma cell lines obtained by screening were amplified in serum-containing complete medium, and the medium was centrifuged and changed to serum-free medium SFM medium to make the cell density 1-2 ⁇ 10 7 /ml, under 5% CO 2 , Cultured at 37°C for 2 weeks, centrifuged to obtain the culture supernatant, purified by Protein G affinity chromatography, and obtained 10 strains of mouse-derived anti-human IL-33 monoclonal antibodies. Named 874F7, 871G1, 864F3, 887B9, 858D5, 868H10, 604A8, 604A12, 646F8, 651H2, respectively.
  • the binding ability of murine antibodies to human IL-33 was determined by indirect enzyme-linked immunosorbent assay.
  • the specific method is as follows: pre-coated with biotin-avidin (SA) and diluted to 2 ⁇ g/ml with coating solution (50 mM carbonate coating buffer, pH 9.6) to coat the plate at 4°C overnight; then use 5% Blocked with nonfat dry milk at 37 °C for 2 hr. Store at -80°C until use.
  • Biotinylated recombinant human IL-33-his protein was diluted to 0.5 ⁇ g/ml with coating solution (50 mM carbonate coating buffer, pH 9.6), and 100 ⁇ l/well was added to the pre-coated SA plate , 37°C, 0.5 hours.
  • the plate was washed three times with PBST, and the antibody to be tested, which was serially diluted with 1% BSA, was sequentially added to the blocked ELISA plate, 100 ⁇ l/well, and placed at 37° C. for 1 h. Wash the plate 3 times with PBST, add HRP-labeled goat anti-mouse IgG secondary antibody, and place at 37°C for 30 min; after washing the plate 3 times with PBST, pat the remaining droplets dry on absorbent paper as much as possible, and add 100 ⁇ l of TMB to each well, at room temperature ( 20 ⁇ 5°C) in the dark for 5 min; add 50 ⁇ l of 2M H 2 SO 4 stop solution to each well to stop the substrate reaction, read the OD value at 450 nm of the microplate reader, and analyze the difference between the antibody to be tested and the target antigen human IL-33-his. binding ability.
  • the results are shown in Figures 1A and 1B, and the antibodies shown have good binding activity.
  • the EC 50 of 874F7, 871G1, 864F3, 887B9, 858D5, and 868H10 antibodies were 0.11 nM, 0.14 nM, 0.27 nM, 0.07 nM, 0.18 nM, and 0.36 nM, respectively.
  • the EC 50 of 604A8, 604A12, 646F8, and 651H2 antibodies were 0.11 nM, 0.16 nM, 0.10 nM, and 0.13 nM, respectively.
  • ST2 is the only receptor of IL-33 that has been discovered so far.
  • IL-33 as a cytokine, relies on ST2 signaling pathway to exert its physiological function, and the blocking activity of the antibody can reflect its neutralizing activity.
  • the specific method is as follows: coat the plate with 2 ⁇ g/ml ST2PBS solution, overnight at 4°C; wash 3 times with PBST, then block with 5% nonfat dry milk, 37°C for 2 hours; wash 3 times with PBST before use; 20ng diluted with 1% BSA /ml IL-33-his-biotin antigen was mixed with 1:1 equal volume of the antibody to be tested in 1% BSA, and incubated at 37°C for one hour; this mixture was added to the pre-coated ST2 plate, and incubated at 37°C for 1 hour.
  • the results are shown in Figures 2A and 2B.
  • the antibodies 874F7, 871G1, 864F3, 887B9, 858D5, and 868H10 shown in Figure 2A all have good blocking activities, with IC 50 of 0.90nM, 0.74nM, 0.23nM, 0.25nM, and 0.16, respectively. nM, 0.27nM.
  • Antibodies 604A8, 604A12, 646F8, 651H2 shown in Figure 2B had no or very weak blocking activity.
  • the biological activity of anti-IL-33 antibodies was determined using HUVEC cells.
  • the specific method is as follows: culture HUVEC cells in a T75 culture flask, transfer the HUVEC cells to the fifth generation of cells, wash the HUVEC cells in the T75 culture flask with 1 ⁇ PBS, and then digest with 1 ml of trypsin for 10 min. 10ml medium to stop digestion, count, plate, 100 ⁇ l/well, 6000/well. The rest were kept in T75 flasks for further culture and cryopreservation.
  • IL-33-his was diluted with medium to 20ng/ml, the antibody to be tested was diluted from 40 ⁇ g/ml, 8 gradients were diluted 4 times, and 4 wells were made of blank.
  • IL-6 inhibition rate was calculated according to the following formula and analyzed by GraphPad Prism 6 software:
  • Inhibition rate (ODAgIL-33-OD administration)/(ODAgIL-33-OD blank) ⁇ 100%.
  • the IC 50 of the inhibitory activity of 874F7, 871G1, 864F3, 887B9, 858D5, 868H10, 604A8, 604A12, and 651H2 were 0.65nM, 1.66nM, 1.52nM, 4.53nM, 12.25nM, 9.35nM, respectively. , 0.08nM, 0.13nM, 0.04nM.
  • antibodies 604A8, 604A12, and 651H2 had poor maximal inhibition rates.
  • the biological activity of anti-IL-33 antibodies was determined using HUVEC cells.
  • the specific method is as follows: culture HUVEC cells in a T75 culture flask, transfer the HUVEC cells to the fifth generation of cells, wash the HUVEC cells in the T75 culture flask with 1 ⁇ PBS, and then digest with 1 ml of trypsin for 10 min. 10ml medium to stop digestion, count, plate, 100 ⁇ l/well, 6000/well. The rest were kept in T75 flasks for further cultivation and cryopreservation.
  • IL-33-his was diluted with medium to 20ng/ml, the antibody to be tested was diluted from 40 ⁇ g/ml, 8 gradients were diluted 4 times, and 4 wells were made of blank.
  • IL-8 inhibition rate was calculated according to the following formula and analyzed by GraphPad Prism 6 software:
  • Inhibition rate (ODAgIL-33-OD administration)/(ODAgIL-33-OD blank) ⁇ 100%.
  • the results are shown in Figure 4.
  • the activity of antibodies 858D5 and 868H10 is very weak.
  • the IC 50 of antibodies 874F7, 871G1, 864F3 and 887B9 are 0.35nM, 0.16nM, 1.46nM and 8.90nM, respectively.
  • the binding ability of murine antibody to cynomolgus monkey IL-33 was determined by indirect enzyme-linked immunosorbent assay. The specific method is as follows: pre-coated with biotin-avidin (SA) and diluted with coating solution (50mM carbonate coating buffer, pH 9.6) to 2 ⁇ g/ml coated plate at 4°C overnight; % nonfat dry milk was blocked at 37°C for 2 hours. Store at -80°C until use.
  • SA biotin-avidin
  • Biotinylated recombinant cynomolgus IL-33-his protein was diluted to 1 ⁇ g/ml with coating solution (50 mM carbonate coating buffer, pH 9.6), and 100 ⁇ l/well was added to the pre-coated SA plate medium, 37°C, 0.5 hours.
  • the plate was washed three times with PBST, and the antibody to be tested, which was serially diluted with 1% BSA, was sequentially added to the blocked ELISA plate, 100 ⁇ l/well, and placed at 37° C. for 1 h.
  • the results are shown in Figure 5, and the antibodies shown can bind well to the cynomolgus monkey IL-33 protein.
  • the EC 50 of antibodies 874F7, 871G1, 864F3, 887B9, 858D5, 868H10, 604A8, 604A12, 646F8, 651H2 were 0.03nM, 0.04nM, 0.06nM, 0.05nM, 0.02nM, 0.05nM, 0.06nM, 0.06nM, 0.06nM, 0.06nM, respectively. nM, 0.06nM.
  • Intravenous administration of antigen IL-33 to animals can cause changes in the inflammatory response in vivo, and the proliferation of spleen cells leads to an increase in spleen weight.
  • This method can be used to evaluate the activity of antibody drugs to neutralize IL-33 in vivo.
  • the specific method is as follows: Female BALB/C mice, weighing 18-20 g, were randomly divided into 6 groups with 10 animals in each group.
  • the experimental results are shown in Figure 6.
  • the average weight of the spleen in the PBS control group was 86.9 ⁇ 9.4 mg, while the weight gain of the Ag-IL-33 group was significantly 195.7 ⁇ 26.9 mg.
  • the average spleen weights of the 864F3, 871G1, and 874F7 antibody-administered groups were 106.3 ⁇ 24.5 mg, 77.7 ⁇ 17.5 mg, and 89.9 ⁇ 14.7 mg, respectively, all of which had obvious neutralizing activity.
  • the heavy chain variable regions and light chain variable regions of the hybridomas 864F3, 874F7 and 871G1 were obtained by methods related to molecular biology, and chimeric antibodies were further constructed.
  • RNA of the three hybridoma cells 864F3, 874F7 and 871G1 was extracted by Trizol, and the mRNA was reverse transcribed to obtain cDNA. Then, using the cDNA as a template, the heavy chain and light chain degenerate primers of the mouse antibody were used respectively ( ⁇ Antibody Engineering ⁇ Volume 1. Edited by Roland Kontermann and Stefan Dübel, the sequence of the combined primers is from page 323) to carry out PCR, sequence the obtained PCR product and analyze it through the kabat database to confirm that the obtained sequence is the variable region sequence of the mouse antibody .
  • 864F3 has two heavy chain variable region gene sequences, the full length is 363bp, each encodes 121 amino acid residues, the nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 3 respectively, and the amino acid sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 4 are shown; 864F3 light chain variable region gene sequence is 321bp in full length, encoding 107 amino acid residues, the nucleotide sequence is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 5 ID NO: 6.
  • the full length of the 874F7 heavy chain variable region gene sequence is 363 bp, encoding 121 amino acid residues, the nucleotide sequence is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8; 874F7 light chain variable region
  • the full-length gene sequence is 318 bp, encoding 106 amino acid residues, the nucleotide sequence is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10.
  • the full length of the 871G1 heavy chain variable region gene sequence is 363 bp, encoding 121 amino acid residues, the nucleotide sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4; 871G1 light chain variable region
  • the full-length gene sequence is 318 bp, encoding 106 amino acid residues, the nucleotide sequence is shown in SEQ ID NO: 11, and the amino acid sequence is shown in SEQ ID NO: 12.
  • the three preferred murine anti-IL-33 antibodies have high homology to the three H-CDRs and three L-CDRs, in particular all have the same L-CDR2 (SEQ ID No: 22).
  • the resulting hybridoma heavy chain variable region sequences were spliced with the human IgG4 constant region (containing the S228P mutation) (the amino acid sequence is shown in SEQ ID NO: 13), and the light chain variable region sequences were combined with the human kappa chain constant region.
  • Each monoclonal antibody was diluted by 3-fold gradient in BSA, the highest concentration was 10 ⁇ g/mL, 12 gradients were diluted, 100 ⁇ L/well was added to ELISA wells, incubated at room temperature for 1 h, and each sample was made into 2 duplicate wells in parallel, washed 3 times with PBST , Dilute the HRP-anti human IgG Fc secondary antibody with 1% BSA prepared in PBST according to the appropriate ratio, add ELISA well, 100 ⁇ L/well, incubate for 1 h at room temperature, wash 3 times with PBST, add TMB color developing solution, 100 ⁇ L/well, color development When the expected color was reached, the color reaction was terminated with 2M H 2 SO 4 , 70 ⁇ L/well, each reaction solution was shaken evenly, and the OD450nm was measured on a microplate reader, the data was analyzed, and the EC 50 was calculated.
  • the final concentration of the highest antibody concentration is 20 ⁇ g/mL, and the final concentration of IL-33-his-biotin is 5ng/mL
  • wash away the unbound or non-specifically bound primary antibody and use the antibody diluent HRP according to the instructions of the antibody.
  • the labeled streptavidin secondary antibody was diluted to an appropriate concentration, added to the ELISA plate, 100 ⁇ L/well, incubated at room temperature for 1 h, washed 5 times with PBST, patted dry on absorbent paper, removed the excess liquid, and added TMB chromogenic solution, 100 ⁇ L/well, develop the color to a suitable depth, add 2M H 2 SO 4 , 70 ⁇ L/well to stop the color development, and measure its absorbance at a wavelength of 450 nm in a multi-function microplate reader to analyze the data.
  • the experimental results are shown in Figure 8.
  • the IC 50 of the chimeric antibodies 874F7-ch, 871G1-ch, 864F3-ch1, and 864F3-ch2 are 0.634nM, 0.853nM, 45.245nM, and 0.580nM, respectively, indicating that each chimeric antibody is IL -33 blocking antibodies can effectively block the binding of IL-33 to its receptor ST2, and 874F7-ch, 871G1-ch and 864F3-ch2 are better.
  • amino acid sequences of the light chain variable region and heavy chain variable region of each candidate murine antibody were analyzed, and the three antigenic complementarity determining regions (CDRs) and four framework regions (FR) of the murine antibody were determined according to the Kabat rule.
  • amino acid sequence of the 864F3 heavy chain complementarity determining region is
  • HCDR1 NYGVH (SEQ ID NO: 15),
  • HCDR2 VIRAGGSSDYNSALMS (SEQ ID NO: 16),
  • HCDR3 DHYFSNSYGGSPY (SEQ ID NO: 17) or
  • HCDR1 KYGVH (SEQ ID NO: 18),
  • HCDR2 VLRAGGTISYNSALMS (SEQ ID NO: 19),
  • HCDR3 DHYYYSSFGGFAS (SEQ ID NO: 20),
  • amino acid sequence of the light chain complementarity determining region is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethy
  • LCDR1 LASQTIATWLA (SEQ ID NO: 21),
  • LCDR2 AATRLAD (SEQ ID NO: 22) and
  • LCDR3 QQLYNTPYT (SEQ ID NO: 23).
  • amino acid sequence of the 874F7 heavy chain complementarity determining region is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • HCDR1 KYGVH (SEQ ID NO: 18),
  • HCDR2 VLRAGGSTGYNSALMS (SEQ ID NO: 24),
  • HCDR3 DHYYYSSYGGFVY (SEQ ID NO: 25),
  • amino acid sequence of the light chain complementarity determining region is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethy
  • LCDR1 LASQTIGAWLA (SEQ ID NO: 26),
  • LCDR2 AATRLAD (SEQ ID NO: 22) and
  • LCDR3 QQLDSSPYT (SEQ ID NO: 27).
  • amino acid sequence of the 871G1 heavy chain complementarity determining region is
  • HCDR1 KYGVH (SEQ ID NO: 18),
  • HCDR2 VLRAGGTISYNSALMS (SEQ ID NO: 19),
  • HCDR3 DHYYYSSFGGFAS (SEQ ID NO: 20),
  • amino acid sequence of the light chain complementarity determining region is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethy
  • LCDR1 LASQTIGAWLA (SEQ ID NO: 26),
  • LCDR2 AATRLAD (SEQ ID NO: 22) and
  • LCDR3 QQLNSTPYT (SEQ ID NO: 28).
  • the CDR region of the murine antibody was transplanted to the selected humanized template to replace the CDR region of the human template.
  • the heavy chain variable region was recombined with the human IgG4 constant region (containing the S228P mutation), and the light chain variable region was It is recombined with the constant region of human kappa chain, and based on the three-dimensional structure of the antibody, the embedded residues, the residues that have direct interaction with the CDR region, and the conformation of VL and VH of each antibody have an important influence.
  • the residues were backmutated, and finally multiple humanized antibodies were obtained.
  • each humanized antibody and chimeric antibody The corresponding heavy and light chain variable regions and sequences of each humanized antibody and chimeric antibody are shown in Table 1b.
  • the heavy chain and light chain of each humanized antibody were respectively constructed into pcDNA3.4 expression vector, transfected into HEK-293F cells, purified by ProteinA to obtain each humanized antibody, and identified by SDS-PAGE electrophoresis and SEC-HPLC. Correct molecular weight and >95% purity.
  • Example 12 Determination of the binding activity of each humanized antibody to human IL-33 by ELISA
  • the experimental results are shown in Figures 9A to 9D.
  • the EC 50s of the chimeric antibody 864F3-ch2 and the humanized antibodies 864F3-HuG, 864F3-Hu1, and 864F3-Hu2 were 0.169nM, 0.760nM, 0.249nM, and 0.181nM, respectively.
  • the humanized antibodies 864F3-HuG, 864F3-Hu1, 864F3-Hu2 lost more affinity for human IL-33, and the chimeric antibodies 864F3-ch2 and 864F3-Hu3, 864F3-Hu4,
  • the EC50 of 864F3-Hu5 was 0.124nM, 0.138nM, 0.135nM, 0.146nM, respectively, and 864F3-Hu3, 864F3-Hu4 and 864F3-Hu5 had almost no affinity for human IL-33 compared to the chimeric antibody 864F3-ch2. Change.
  • the EC50 of the chimeric antibody 874F7-ch and the humanized antibodies 874F7-H ⁇ g, 874F7-Hul, 874F7-Hu2 were 0.177nM, 0.170nM, 0.129nM, and 0.136nM, respectively.
  • Humanized antibodies 874F7-Hu1 and 874F7-Hu2 showed no loss of affinity for human IL-33 compared to chimeric antibody 874F7-ch.
  • the EC50 of the chimeric antibody 871G1-ch and the humanized antibodies 871G1-HuG, 871G1-Hul, 871G1-Hu2 were 0.255nM, 0.187nM, 0.191nM, and 0.176nM, respectively. None of the humanized antibodies 871G1-Hu1, 871G1-Hu2 and 871G1-HuG lost their affinity for human IL-33 compared to the chimeric antibody 871G1-ch.
  • the experimental results are shown in Figure 10.
  • the IC50 values of the humanized antibodies 864F3-Hu4, 864F3-Hu5, 871G1-Hu1, 871G1-Hu2, 874F7-Hu1, and 874F7-Hu2 for blocking the binding of IL-33 to ST2 protein They were 0.512nM, 0.473nM, 0.404nM, 0.361nM, 0.451nM, and 0.350nM, respectively. It was shown that each humanized antibody retained the blocking effect on the binding of IL-33 to ST2.
  • Each antibody to be tested was recovered using a chip covalently coupled with Protein A/G (purchased from GE Healthcare, product number BR-1005-30).
  • the relevant operating parameters are as follows: antibody concentration 2 ⁇ g/mL, contact time 75s, flow rate 10 ⁇ L /min, the regeneration contact time is 30s.
  • Example 15 Inhibitory effect of humanized antibody on IL-33-induced IL-6 expression in HUVEC cells
  • the activity of each humanized antibody was evaluated by measuring the inhibitory effect of each humanized antibody on IL-33-his protein-induced expression of IL-6 in HUVEC cells.
  • the specific experimental steps are as follows: the HUVEC cells in the T75 culture flask were digested and counted, and then plated in complete medium, 100 ⁇ L/well, 6000 cells/well, and cultured in a 37°C incubator. After 2 hours, the administration started after the cells adhered.
  • IL-33-his Dilute the antibody to be tested with complete medium, the highest concentration is 200nM, 9 gradients of 5-fold dilution, set positive blank control and negative blank control; after dilution, the antibody is mixed with 20ng/mL IL-33-his by 1:1 volume ( The highest concentration of antibody added to cells was 50 nM and the final concentration of IL-33-his was 5 ng/mL). Incubate for 30 min at room temperature; add 100 ⁇ L of the antibody/IL-33-his mixture to the cell solution and mix gently, incubate at 37 °C CO 2 for 20 h, collect the supernatant by centrifugation, and store it at -80 °C for IL-6 determination.
  • Rat-Anti-human IL-6 protein Dilute Rat-Anti-human IL-6 protein to 2.5 ⁇ g/mL with ELISA coating solution, coat ELISA plate, 100 ⁇ L/well, put in a wet box, 4°C, coat for 16h; wash ELISA plate three times with PBST, Remove unbound proteins, pat the ELISA plate dry on absorbent paper, remove excess liquid, then block with 2% BSA prepared in PBS, 200 ⁇ L/well at room temperature for 1-2 h; use 1% BSA prepared in PBS according to 3
  • the IL-6 standard was serially diluted to a maximum concentration of 30ng/mL.
  • the experimental results are shown in Figure 11.
  • the humanized antibodies 864F3-Hu4, 864F3-Hu5, 871G1-Hu1, 871G1-Hu2, 874F7-Hu1, and 874F7-Hu2 inhibit the IL-33-induced IL-6 expression in HUVEC cells IC
  • the 50 values were 0.073nM, 0.147nM, 0.210nM, 0.175nM, 0.051nM, 0.050nM, respectively. It shows that each humanized antibody can effectively inhibit the secretion of IL-6 induced by IL-33-his protein in HUVEC cells, especially 874F7-Hu1 and 874F7-Hu2 have relatively better effects.
  • Example 16 Inhibitory effect of humanized antibody on IL-33-induced IFN ⁇ secretion by PBMC
  • each humanized antibody was evaluated by measuring the inhibitory effect of each humanized antibody on IL-33-his protein-induced PBMC secretion of IFN ⁇ .
  • the specific experimental steps are as follows: centrifuge and count fresh PBMC, wash once with PBS, and dilute to 4 ⁇ 10 6 cells/Ml with RPMI1640 complete medium; add PBMC cells to a 96-well U-shaped cell culture plate, and add 50 ⁇ L (2 ⁇ 10 5 cells/well), placed in a 37°C incubator to recover; dilute the antibody with RPMI1640 complete medium, the highest concentration is 200nM (final concentration is 50nM), follow a 3-fold gradient dilution, and add an equal volume of 40ng/mL IL to each well after dilution -33-his (final concentration of 10ng/mL), incubate at 37°C for 30min; add 50 ⁇ L of IL-12 diluted in 40ng/mL complete medium to the above PBMC cell culture plate, and then add 100 ⁇ L
  • the experimental results are shown in Figure 12.
  • the IC 50 values of the humanized antibodies 864F3-Hu4, 864F3-Hu5, 871G1-Hu1, 871G1-Hu2, 874F7-Hu1, and 874F7-Hu2 on IL-33-induced IFN ⁇ secretion by PBMC were respectively were 1.224nM, 0.839nM, 1.395nM, 1.061nM, 0.850nM, 1.736nM. It shows that each humanized antibody can effectively inhibit IL-33-his protein-induced human PBMC to secrete IFN ⁇ , and the activity of 874F7-Hu2 is relatively poor.
  • Example 17 Inhibitory effect of humanized antibody on IL-33-induced NK cell secretion of IFN ⁇
  • NK cells were isolated according to the instructions of Nk Cell Isolation Kit human (purchased from Miltenyi, Cat. No. 130-092-657). NK cells were counted after washing twice with PBS, diluted to 0.8 ⁇ 10 6 cells/mL with 1640+10% FBS+1% Glutamax medium, and plated in 96-well cell culture plates with 50 ⁇ L of cell suspension per well. Each antibody was diluted with 1640+10% FBS+1% Glutamax medium, the final maximum effect concentration was 100 nM, 1/4 serial dilution. After dilution, IL-33-his with a final concentration of 10 ng/mL was added to each well and incubated at 37 °C for 30 min.
  • IL-12 with a final concentration of 2 ng/mL diluted in complete medium was added to the 96-well cell culture plate containing NK cells, and finally 100 ⁇ L of the mixture of antibody and IL-33 was added, and incubated at 37°C for 24 hours. After that, the cell culture supernatant was collected to measure the secretion level of IFN ⁇ .
  • Example 16 for the detection method of IFN ⁇ .
  • the experimental results are shown in Figure 13.
  • Humanized antibodies 864F3-Hu4, 864F3-Hu5, 871G1-Hu1, 871G1-Hu2, 874F7-Hu1, 874F7-Hu2 have inhibitory effects on IL-33-induced NK cells to secrete IFN ⁇ , IC 50
  • the values were 0.904nM, 1.021nM, 0.851nM, 0.900nM, 0.742nM, 1.559nM, respectively.
  • Example 18 Inhibitory effect of humanized antibody on IL-33-induced secretion of IL-5 and IL-13 by KU812 cells
  • the basophilic leukemia cells KU812 in logarithmic growth phase were collected, centrifuged, counted, and treated with IMDM containing 20% FBS.
  • the medium complete medium, purchased from Gibco, Cat. No. 11965-092
  • the medium was resuspended, and 96-well cell culture plates were plated at 20,000 cells per well.
  • Each antibody to be tested was prepared in complete medium, and the administration group was diluted by 3-fold gradient. Each group was serially diluted with 9 gradients. The maximum working concentration of the antibody was 200 ⁇ g/mL.
  • IL-33-his was prepared in complete medium.
  • the concentration was 700ng/ml. After mixing at a 1:1 ratio, it was added to the above-mentioned 96-well cell culture plate, and the final volume of each well was 200 ⁇ L.
  • the untreated group was set as the negative control, and only IL-33 was added as the single-drug control group.
  • Two duplicate wells were made in parallel for each concentration, and the cells were cultured in a 37°C cell incubator for 48 hours. The supernatant was collected for the determination of IL-5 and IL-13 secretion.
  • the hu-IL-13 standard was diluted with 1% BSA prepared in PBST according to a 3-fold gradient, the highest concentration was 10ng/mL, diluted in 12 gradients, added to ELISA wells, 100 ⁇ L/well, incubated at room temperature for 2h, and each sample was prepared in parallel. 2 duplicate holes. Add 50 ⁇ L/well of collected cell supernatant. Wash away the unbound or non-specifically bound primary antibody, and use the antibody diluent to dilute Biotin-anti-human-IL-5 (purchased from BD, Cat. No. 554491) and Biotin-anti-human-IL-13 with antibody diluent according to the instructions of the antibody.
  • the secondary antibody was diluted to an appropriate concentration, added to the ELISA plate, 100 ⁇ L/well, and incubated at room temperature for 2 h. Wash three times with PBST, pat the ELISA plate dry on absorbent paper, remove excess liquid, dilute HRP-labeled Streptavidin (purchased from BD, Cat. No. 554066) to the appropriate concentration with antibody diluent, add to the ELISA plate, 100 ⁇ L/well , incubated at room temperature for 0.5h.
  • the experimental results are shown in Figure 14.
  • the humanized antibodies 864F3-Hu4 and 874F7-Hu1 can effectively inhibit IL-33-induced KU812 cells to secrete IL-5 with IC 50 of 27.605nM and 7.828nM, respectively.
  • humanized antibodies 864F3-Hu4 and 874F7-Hu1 can also effectively inhibit IL-33-induced IL-13 secretion by KU812 cells, with IC50 of 14.745nM and 5.219nM, respectively.
  • the experimental method refers to Example 14, wherein the antigens are respectively cynomolgus monkey IL-33 protein (purchased from Sino biological, item number 90912-CNAE) and mouse IL-33 protein (purchased from R&D, 3626-ML-010/CF), At the same time, human IL-33 protein was set as a control.
  • Table 3 Both 864F3-Hu4 and 874F7-Hu1 cross-react with cynomolgus monkey IL-33 protein, and are consistent with the binding characteristics of each antibody and human IL-33 protein. In addition, 864F3-Hu4 and 874F7-Hu1 also had certain cross-reactivity with mouse IL-33 protein, but their affinity was significantly lower than the binding of each antibody to human IL-33 protein.
  • each experimental group had 10 BALB/C mice.
  • the results showed that the spleen in the IL-33 antigen group had a significant weight gain of 176.49 mg compared with the control group (spleen weight: 80.55 mg).
  • the mean spleen weights of the 864F3-Hu4, 864F3-Hu5, 871G1-Hu1, 871G1-Hu2, 874F7-Hu1, and 874F7-Hu2 antibody-administered groups were 78.39 mg, 86.6 mg, 92.82 mg, 98.87 mg, 75.63 mg, and 77.49 mg, respectively mg, it can be seen that each humanized antibody has obvious neutralizing activity in vivo.
  • the experimental method refers to Example 7.
  • the experimental results are shown in Figure 17.
  • After intraperitoneal injection of human IL-33 it can significantly stimulate the spleen of mice (IL-33 group), and the spleen weight is about 194.6 mg.
  • Stimulated with human IL-33) was 76.1 mg, and when 5 mg/kg of 864F3-Hu4 and 874F7-Hu1 were administered as a single treatment, it could effectively inhibit spleen enlargement in mice, and the spleen weights were 65.4 mg and 55.9 mg, respectively.
  • mice in each group were euthanized, and the levels of eosinophils and IL-5 in peripheral blood were detected respectively.
  • a part of the whole blood of the above mice was taken out and placed in an ordinary EP tube, and placed at 4°C for more than 5 hours to allow complete coagulation. After coagulation, the EP tube was placed in a centrifuge pre-cooled at 4°C and centrifuged at 8000 rmp for 6 min. After centrifugation, serum was taken out, placed in a new centrifuge tube, and centrifuged again at 8000 rmp for 6 min in a centrifuge pre-cooled at 4°C. After centrifugation, the supernatant was removed, aliquoted, and stored at -80.
  • the standard and test serum samples were diluted according to the instructions of the BD CBA Mouse Enhanced Sensivity Master Buffer Kit (purchased from BD biosciences, Cat. No. 562246). Subsequently, each sample was processed according to the instructions of the Mouse IL-5 Enhanced Sensitivity Flex Set (purchased from BD biosciences, item number 562234), and the detection parameters were set to detect each sample on a flow meter (BD FACSCelesta). The collected data were analyzed, and the content of IL-5 in serum samples of each experimental group was calculated according to the standard curve.
  • mice A part of the whole blood of the above mice was taken out and placed in an EDTA anticoagulation tube, upside down evenly, and placed at 4°C for later use. Take 10 mL round-bottom centrifuge tubes and add 2 ⁇ L mouse Fc blocker (purchased from BD biosciences, Cat. No. 553142) to each tube. Take 50 ⁇ L of blood sample for each sample into a 10mL centrifuge tube, and gently pipette to mix the blood sample with the blocker evenly. Incubate on ice for 15min. To each centrifuge tube, add 2 ⁇ L of the three mixed antibodies: 0.5 ⁇ L CD45.2 Monoclonal Antibody (104), PerCP-Cyanine5.5 (purchased from eBioscience TM , Cat. No.
  • CD170 Monoclonal Antibody (1RNM44N), PE (purchased from eBioscience TM , Cat. No. 12-1702-82), 1 ⁇ L Ly-6G/Ly-6C Monoclonal Antibody (RB6-8C5), APC-eFluor 780 (purchased from eBioscience TM , Cat. No. 47-5931-82); incubate on ice for 1 h in the dark. Add 500 mL of 1x Lysing buffer (purchased from BD Biosciences, Cat. No. 555899) to the centrifuge tube, mix well, and lyse the red blood cells at room temperature for 5 min.
  • 1x Lysing buffer purchased from BD Biosciences, Cat. No. 555899
  • the centrifuge tube was centrifuged at 350 g for 5 min. The supernatant was discarded, 3 mL of pre-cooled PBS was added to the tube to wash the cells, and the cells were centrifuged at 350 g for 5 min. Discard the supernatant, add 500 ⁇ L of pre-chilled, filter-treated PBS to the tube, and gently bounce the settled cells. The cell suspension was filtered through a cell mesh into a flow tube, and the ratio of eosinophils to leukocytes in each sample was analyzed by flow cytometry.
  • biotin-avidin (SA) was pre-diluted with coating solution (50 mM carbonate coating buffer, pH 9.6) to 2 ⁇ g/mL, 100 ⁇ L/well, coated with ELISA plate, 4°C, overnight; washed 3 times with PBST, then blocked with 2% BSA prepared in PBS for 2 h at room temperature, 200 ⁇ L/well. After washing once with PBST, 864F3 and 874F7 were added at a final concentration of 10 ⁇ g/mL, 100 ⁇ L/well, and incubated at room temperature for 1 h.
  • coating solution 50 mM carbonate coating buffer, pH 9.6
  • BSA 2% BSA prepared in PBS for 2 h at room temperature, 200 ⁇ L/well.
  • 864F3 and 874F7 were added at a final concentration of 10 ⁇ g/mL, 100 ⁇ L/well, and incubated at room temperature for 1 h.
  • the "KKDEKKDKVLLSYYESQHPNESGDGVDGK” region (position 41-70 of SEQ ID NO.55) was subjected to alanine-scanning site-directed mutagenesis by PCR (polymerase chain reaction), followed by prokaryotic expression and purification to obtain the following amino acid site mutations respectively IL-33 mutant protein:
  • FIG. 24 The positions of the above sites in the 3D structure diagram of IL-33 (derived from PDB: 2KLL) are shown in Figure 24, which further illustrates that the binding epitope of 874F7-Hu1 and IL-33 includes the above-mentioned key amino acid sites in Linear and spatial epitopes within.

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Abstract

本发明涉及一种结合人IL-33的抗体或其抗原结合片段，能够以高亲和力方式结合人IL-33，阻断ST2与IL-33的结合，应用于制备治疗IL-33高表达的疾病（例如哮喘、特异反应性/过敏性皮炎、慢性鼻窦炎、慢性阻塞性肺病(COPD)等）的药物，具有良好的临床应用前景。

Description

结合人IL-33的抗体、其制备方法和用途 技术领域
本发明属于生物技术领域，涉及一种结合人IL-33的抗体、其制备方法和用途。
背景技术
白介素33(IL-33)是一个多功能细胞因子，属于IL-1家族新成员。由IL-33基因编码，在结构细胞如平滑肌细胞、上皮细胞和内皮细胞中组成性表达，在巨噬细胞和树突状细胞中，IL-33可以由炎症因子诱导表达。研究发现，IL-33是一种双功能蛋白。一方面，IL-33定位于细胞核内，发挥转录因子的作用；另一方面，IL-33分泌到细胞外，通过与其受体ST2相互作用发挥细胞因子的作用。作为细胞因子，IL-33是一种TH-2型的细胞因子，被认为作为警报素，通过结合ST2，诱导TH-2细胞分泌IL-4、IL-5和IL-13等TH-2型的细胞因子。此外，IL-33还可引起肥大细胞、嗜碱性粒细胞分泌炎性细胞因子和趋化因子，例如IL-1β、IL-6、IL-8、TNFα等，导致NK细胞和NKT细胞分泌TH-1型细胞因子，如IFNγ等。
哮喘又名支气管哮喘。支气管哮喘是由多种细胞及细胞组分参与的慢性气道炎症。哮喘一直被认为是CD4+Th-2细胞驱动的气道炎症。然而，抗CD4抗体几乎完全耗尽CD4+细胞，但并不能完全减少哮喘小鼠肺中IL-4、IL-5或IL-13的产生，这表明这些Th-2细胞因子的其他细胞来源肯定存在。IL-33能从表达ST2的ILC2s中产生IL-5和IL-13，这表明即使在没有Th-2细胞的情况下，也可以诱导IL-5诱导的嗜酸性粒细胞增多和IL-13诱导的粘液产生。此外，IL-33在哮喘患者肺中的表达水平高于健康者，IL-33在重症哮喘患者肺组织中的表达尤为明显。IL-33促进重症哮喘患儿哮喘成纤维细胞胶原合成，提示IL-33在重症哮喘特征性气道重塑的发生发展中起一定作用。过敏性气道炎症可通过抗IL-33抗体治疗而减轻。由于IL-33能够激活表达ST2的Th-2细胞，Th-2细胞和ILC2细胞产生的IL-5和IL-13参与了哮喘的发病过程。这些发现表明IL-33可以协调先天免疫和后天免疫之间的桥梁，从而发展为严重哮喘表型。除哮喘外， IL-33途径还涉及多种病症治疗，如特异反应性/过敏性皮炎、关节炎、慢性鼻窦炎、慢性阻塞性肺病(COPD)、系统性硬化症、肝纤维化、牛皮癣、溃疡性结肠炎、克罗恩氏病、多发性硬化症、糖尿病肾脏疾病、发炎性肠道疾病、银屑病、嗜酸性食管炎、糖尿病性黄斑水肿、年龄相关性黄斑变性、干眼病、肿瘤等。本发明提供了以高亲和力方式结合IL-33并有效中和IL-33活性的IL-33抑制剂。
发明内容
为了解决上述技术问题，本发明的发明人进行了大量试验，从抗原免疫、杂交瘤筛选、抗体表达纯化到生物活性鉴定，筛选获得了特异性结合人IL-33的鼠源抗体，并在此基础上，进一步构建获得其嵌合抗体以及人源化抗体。
因此，本发明的目的在于提供一种结合人IL-33的抗体或其抗原结合片段；提供编码所述结合人IL-33的抗体或其抗原结合片段的核苷酸分子；提供包含所述核苷酸分子的表达载体；提供所述表达载体的宿主细胞；提供所述结合人IL-33的抗体或其抗原结合片段制备方法；提供包含所述结合人IL-33的抗体或其抗原结合片段药物组合物；提供所述结合人IL-33的抗体或其抗原结合片段在制备药物中的应用。
为了实现上述目的，本发明采用了如下技术方案：
本发明一方面提供一种结合人IL-33的抗体或其抗原结合片段，其中该抗体或其抗原结合片段与人IL-33结合的亲和力EC 50小于1nM。
在另一优选例中，所述抗体的轻链具有SEQ ID No:22所示的L-CDR2，并且具有以下特征：
(t1)阻断IL-33和受体ST2的结合；
(t2)抑制IL-33诱导的HUVEC细胞的IL-6分泌；
(t3)抑制IL-33蛋白诱导人PBMC分泌IFNγ；
(t4)抑制IL-33诱导NK细胞分泌IFNγ；和
(t5)抑制IL-33诱导KU812细胞分泌IL-5和IL13。
在另一优选例中，所述抗体对人IL33的Kd值远远小于对小鼠IL33的Kd值(相差20倍或更多)。
在另一优选例中，所述抗体包括：
(a)重链互补决定区H-CDR1、H-CDR2、H-CDR3，所述H-CDR1如SEQ ID NO：18所示或SEQ ID NO：18至多2个氨基酸替换突变的突变体；H-CDR2如SEQ ID NO：19所示或SEQ ID NO：19至多4个氨基酸替换突变的突变体；H-CDR3的氨基酸序列如SEQ ID NO：20所示或SEQ ID NO：20至多7个氨基酸替换突变的突变体，和
(b)轻链互补决定区L-CDR1、L-CDR2、L-CDR3，所述L-CDR1的氨基酸序列如SEQ ID NO：21所示或SEQ ID NO：21至多2个氨基酸替换突变的突变体，所述L-CDR2的氨基酸序列如SEQ ID NO：22所示、所述L-CDR3的氨基酸序列如SEQ ID NO：23所示或SEQ ID NO：23至多3个氨基酸替换突变的突变体。作为优选的方案，所述结合人IL-33的抗体或其抗原结合片段，包括：
(a)重链互补决定区H-CDR1、H-CDR2、H-CDR3，所述H-CDR1、H-CDR2、H-CDR3的氨基酸序列分别如SEQ ID NO：15、16和17所示，或者分别如SEQ ID NO：18、19和20所示，或者分别如SEQ ID NO：18、24和25所示，和
(b)轻链互补决定区L-CDR1、L-CDR2、L-CDR3，所述L-CDR1、L-CDR2、L-CDR3的氨基酸序列分别如SEQ ID NO：21、22和23所示，或者分别如SEQ ID NO：26、22和27所示，或者分别如SEQ ID NO：26、22和28所示。
在另一优选例中，所述的结合人IL-33的抗体或其抗原结合片段，包括：
(a1)重链互补决定区H-CDR1、H-CDR2、H-CDR3，所述H-CDR1、H-CDR2、H-CDR3的氨基酸序列分别如SEQ ID NO：15、16和17所示，和
(b1)轻链互补决定区L-CDR1、L-CDR2、L-CDR3，所述L-CDR1、L-CDR2、L-CDR3的氨基酸序列分别如SEQ ID NO：21、22和23所示；或
(a2)重链互补决定区H-CDR1、H-CDR2、H-CDR3，所述H-CDR1、H-CDR2、H-CDR3的氨基酸序列分别如18、19和20所示，和
(b2)轻链互补决定区L-CDR1、L-CDR2、L-CDR3，所述L-CDR1、L-CDR2、L-CDR3的氨基酸序列分别如SEQ ID NO：21、22和23所示；或
(a3)重链互补决定区H-CDR1、H-CDR2、H-CDR3，所述H-CDR1、H-CDR2、H-CDR3的氨基酸序列分别如SEQ ID NO：18、24和25所示，和
(b3)轻链互补决定区L-CDR1、L-CDR2、L-CDR3，所述L-CDR1、L-CDR2、L-CDR3的氨基酸序列分别如SEQ ID NO：26、22和27；或
(a4)重链互补决定区H-CDR1、H-CDR2、H-CDR3，所述H-CDR1、H-CDR2、H-CDR3的氨基酸序列分别如SEQ ID NO：18、19和20所示，和
(b4)轻链互补决定区L-CDR1、L-CDR2、L-CDR3，所述L-CDR1、L-CDR2、L-CDR3的氨基酸序列分别如SEQ ID NO：26、22和28所示。
在另一优选例中，所述的结合人IL-33的抗体或其抗原结合片段包括：
(a2)重链互补决定区H-CDR1、H-CDR2、H-CDR3，所述H-CDR1、H-CDR2、H-CDR3的氨基酸序列分别如18、19和20所示，和
(b2)轻链互补决定区L-CDR1、L-CDR2、L-CDR3，所述L-CDR1、L-CDR2、L-CDR3的氨基酸序列分别如SEQ ID NO：21、22和23所示；或
(a3)重链互补决定区H-CDR1、H-CDR2、H-CDR3，所述H-CDR1、H-CDR2、H-CDR3的氨基酸序列分别如SEQ ID NO：18、24和25所示，和
(b3)轻链互补决定区L-CDR1、L-CDR2、L-CDR3，所述L-CDR1、L-CDR2、L-CDR3的氨基酸序列分别如SEQ ID NO：26、22和27。
在另一优选例中，上述任一CDR的氨基酸序列中包含经过添加、缺失、修饰和/或取代1、2、3、4、5、6或7个氨基酸的衍生CDR序列，并且使得含有所述衍生CDR序列的VH和VL所构成的衍生抗体能够保留与IL-33结合的亲和力。
本发明“抗体(Ab)”是约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。本发明的抗体包括单克隆抗体、多克隆抗体、由至少两种抗体形成的多特异性抗体(例如双特异性抗体)等。
本发明“单克隆抗体”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤培养来合成的，不会被 其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本发明“抗原结合片段”是指能够与人IL-33特异性结合的抗体的片段。本发明的抗原结合片段的例子包括Fab片段、F(ab’) 2片段、Fv片段等。Fab片段是用木瓜蛋白酶消化抗体产生的片段。F(ab’) 2片段是用胃蛋白酶消化抗体产生的片段。Fv片段是由抗体的重链可变区和轻链可变区紧密非共价关联的二聚物组成。
作为优选的方案，所述的抗体为鼠源抗体、嵌合抗体或人源化抗体。
本发明“鼠源抗体”是指来源于大鼠或小鼠的抗体，优选小鼠。本发明的鼠源抗体为使用人IL-33为抗原免疫小鼠并进行杂交瘤细胞筛选获得。
本发明“嵌合抗体”是指包含来源于一个物种的重链和轻链可变区序列以及来源于另一个物种的恒定区序列的抗体，例如具有与人恒定区连接的鼠重链和轻链可变区的抗体。优选的，本发明的嵌合抗体是由鼠源抗体864F3、874F7、871G1重链可变区再与包含突变的人IgG1、IgG2、IgG3或IgG4重链恒定区重组，轻链可变区与人的kappa链恒定区重组获得。
本发明“人源化抗体”是指其CDR来源于非人物种(优选小鼠)抗体，抗体分子中残余的部分(包括框架区和恒定区)来源于人抗体。此外，框架区残基可被改变以维持结合亲和性。优选的，本发明的人源化抗体由鼠源抗体864F3、874F7、871G1的CDR区和来源自人抗体的非CDR区重组，重链可变区再与包含突变的人IgG1、IgG2、IgG3或IgG4重链恒定区重组，轻链可变区与人的kappa链恒定区重组，并对部分有重要影响的残基进行突变获得。
作为优选的方案，所述的抗原结合片段包括Fab片段、F(ab’) 2片段、Fv片段。
作为优选的方案，所述的结合人IL-33的抗体或其抗原结合片段的重链可变区和轻链可变区的氨基酸序列分别如SEQ ID NO：2和SEQ ID NO：6所示，或者分别如SEQ ID NO：4和SEQ ID NO：6所示，或者分别如SEQ ID NO：8和SEQ ID NO：10所示，或者分别如SEQ ID NO：4和SEQ ID NO：12所示。
作为优选的方案，所述的结合人IL-33的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：32所示，重链可变区的氨基酸序列如SEQ ID NO：29、30、31、33、34或35所示。
作为优选的方案，所述的结合人IL-33的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：40所示，重链可变区的氨基酸序列如SEQ ID NO：37、38或39所示。
作为优选的方案，所述抗体的重链恒定区选自人IgG1、IgG2、IgG3或IgG4的重链恒定区。
作为优选的方案，所述的结合人IL-33的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：36所示，重链可变区的氨基酸序列如SEQ ID NO：33、34或35所示。
在另一优选例中，所述的结合人IL-33的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：32所示，重链可变区的氨基酸序列如SEQ ID NO：34所示；或所述的结合人IL-33的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：40所示，重链可变区的氨基酸序列如SEQ ID NO：38所示。在另一优选例中，所述轻链可变区的氨基酸序列与如序列表中SEQ ID NO：32、40或36所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在另一优选例中，所述重链可变区的氨基酸序列与如序列表中SEQ ID NO：29、30、31、33、34、35、37、38或39所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
作为优选的方案，所述的结合人IL-33的抗体或其抗原结合片段的重链恒定区和轻链恒定区的氨基酸序列分别如SEQ ID NO：13和SEQ ID NO：14所示。
在另一优选例中，所述的结合人IL-33的抗体或其抗原结合片段与IL-33蛋白的结合表位包含对应于SEQ ID NO.55的选自下组的位点：
第45位赖氨酸(K45)、第49位缬氨酸(V49)、第65位天冬氨酸(D65)、第50位亮氨酸(L50)、第60位丝氨酸(S60)、第52位丝氨酸(S52)、第 48位赖氨酸(K48)、第51位亮氨酸(L51)、第53位酪氨酸(Y53)、第55位谷氨酸(E55)；
更佳地，包含选自下组的位点：第45位赖氨酸(K45)、第49位缬氨酸(V49)、第65位天冬氨酸(D65)、第50位亮氨酸(L50)位点。
在另一优选例中，所述位点对应于野生型人IL-33蛋白的氨基酸序列，所述氨基酸序列为NCBI:NP_254274.1的第112位Ser至270位Thr。
本发明另一方面提供了一种核苷酸分子，所述核苷酸分子编码上述结合人IL-33的抗体或其抗原结合片段。
作为优选的方案，所述核苷酸分子编码重链可变区和轻链可变区的核苷酸序列分别如SEQ ID NO：1和SEQ ID NO：5所示所示，或者分别如SEQ ID NO：3和SEQ ID NO：5所示，或者分别如SEQ ID NO：7和SEQ ID NO：9所示，或者分别如SEQ ID NO：3和SEQ ID NO：11所示。
作为优选的方案，所述核苷酸分子编码重链可变区的核苷酸序列分别如SEQ ID NO：41、42、43、45、46或47所示，编码轻链可变区的核苷酸序列如SEQ ID NO：44所示。
作为优选的方案，所述核苷酸分子编码重链可变区的核苷酸序列分别如SEQ ID NO：49、50或51所示，编码轻链可变区的核苷酸序列如SEQ ID NO：52所示。
作为优选的方案，所述核苷酸分子编码重链可变区的核苷酸序列分别如SEQ ID NO：45、46或47所示，编码轻链可变区的核苷酸序列如SEQ ID NO：48所示。
本发明所述核苷酸分子的制备方法为本领域常规的制备方法，较佳地包括以下制备方法：通过基因克隆技术例如PCR方法等，获得编码上述单克隆抗体的核苷酸分子，或者通过人工全序列合成的方法得到编码上述单克隆抗体的核苷酸分子。
本领域技术人员知晓，编码上述结合人IL-33的抗体或其抗原结合片段的氨基酸序列的核苷酸序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该结合人 IL-33的抗体或其抗原结合片段基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明另一方面提供了一种表达载体，所述表达载体含有上述的核苷酸分子。
其中所述表达载体为本领域常规的表达载体，是指包含适当的调控序列，例如启动子序列、终止子序列、多腺苷酰化序列、增强子序列、标记基因和/或序列以及其他适当的序列的表达载体。所述表达载体可以是病毒或质粒，如适当的噬菌体或者噬菌粒，更多技术细节请参见例如Sambrook等，Molecular Cloning：A Laboratory Manual，第二版，Cold Spring Harbor Laboratory Press，1989。许多用于核酸操作的已知技术和方案请参见Current Protocols in Molecular Biology，第二版，Ausubel等编著。本发明所述表达载体较佳地为pDR1，pcDNA3.1(+)，pcDNA3.1/ZEO(+)，pDHFR，pcDNA4，pDHFF，pGM-CSF或pCHO 1.0。
本发明另外提供了一种宿主细胞，所述宿主细胞含有上述的表达载体。
本发明所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核苷酸可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞，所述宿主细胞较佳地包括：COS、CHO(中国仓鼠卵巢，Chinese H amster Ovary)、NS0、sf9、sf21、DH5α、BL21(DE3)或TG1，更佳地为E.coli TG1、BL21(DE3)细胞(表达单链抗体或Fab抗体)或者CHO-K1细胞(表达全长IgG抗体)。将前述表达载体转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
本发明另一方面提供了上述的结合人IL-33的抗体或其抗原结合片段的方法，其特征在于，所述方法包括以下步骤：
a)在表达条件下，培养上述的宿主细胞，从而表达所述的结合人IL-33的抗体或其抗原结合片段；
b)分离并纯化a)所述的结合人IL-33的抗体或其抗原结合片段。
本发明所述的宿主细胞的培养方法、所述抗体的分离和纯化方法为本领域常规方法，具体操作方法请参考相应的细胞培养技术手册以及抗体分离纯化技术手册。本发明中公开的结合人IL-33的抗体或其抗原结合片段的制备方法包括：在 表达条件下，培养上述的宿主细胞，从而表达所述的结合人IL-33的抗体或其抗原结合片段；分离和纯化所述的所述的结合人IL-33的抗体或其抗原结合片段。利用上述方法，可以将重组蛋白纯化为基本均一的物质，例如在SDS-PAGE电泳上为单一条带。
可以利用亲和层析的方法对本发明公开的所述的结合人IL-33的抗体或其抗原结合片段进行分离纯化，根据所利用的亲和柱的特性，可以使用常规的方法例如高盐缓冲液、改变PH等方法洗脱结合在亲和柱上的所述的结合人IL-33的抗体或其抗原结合片段。本发明的发明人对所得所述的结合人IL-33的抗体或其抗原结合片段进行了检测实验，实验结果表明该所述的结合人IL-33的抗体或其抗原结合片段能很好地与抗原结合，具有较高的亲和力。
本发明另一方面提供了一种组合物，所述组合物含有上述的结合人IL-33的抗体或其抗原结合片段和药学上可接受的载体。
本发明提供的结合人IL-33的抗体或其抗原结合片段，可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的结合人IL-33的抗体或其抗原结合片段的构像完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。通常情况下，对于液体制剂，通常可以在2℃-8℃条件下保存至少稳定一年，对于冻干制剂，在30℃至少六个月保持稳定。所述双特异性抗体制剂可为制药领域常用的混悬、水针、冻干等制剂。
对于本发明公开的结合人IL-33的抗体或其抗原结合片段水针或冻干制剂，药学上可以接受的载体较佳地包括但不限于：表面活性剂、溶液稳定剂、等渗调节剂和缓冲液之一或其组合。其中表面活性剂较佳地包括但不限于：非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或80)；poloxamer(如poloxamer 188)；Triton；十二烷基硫酸钠(SDS)；月桂硫酸钠；十四烷基、亚油基或十八烷基肌氨酸；Pluronics；MONAQUATTM等，其加入量应使结合人IL-33的抗体或其抗原结合片段的颗粒化趋势最小。溶液稳定剂较佳地包括但不限于以下列举之一或其组合：糖类，例如，还原性糖和非还原性糖；氨基酸类，例如，谷氨酸单钠或组氨酸；醇类，例如：三元醇、高级糖醇、丙二醇、聚乙二醇等，溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到稳定的时间内保持稳定状态。等渗调节剂较佳地包括但不限于氯化钠、甘露醇之一或其 组合。缓冲液较佳地包括但不限于：Tris、组氨酸缓冲液、磷酸盐缓冲液之一或其组合。
本发明另一方面提供了一种抗体药物偶联物，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分包含上述的结合人IL-33的抗体或其抗原结合片段；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
本发明另一方面提供了上述的结合人IL-33的抗体或其抗原结合片段或药物组合物、抗体药物偶联物在制备治疗哮喘、关节炎、特异反应性/过敏性皮炎、慢性鼻窦炎、慢性阻塞性肺病(COPD)、系统性硬化症、肝纤维化、牛皮癣、溃疡性结肠炎、克罗恩氏病、多发性硬化症、糖尿病肾脏疾病、发炎性肠道疾病、银屑病、嗜酸性食管炎、糖尿病性黄斑水肿、年龄相关性黄斑变性、干眼病、肿瘤的药物中的应用。所述关节炎包括类风湿关节炎、骨关节炎、强直性脊柱炎、痛风性关节炎、反应性关节炎、感染性关节炎、创伤性关节炎、银屑病关节炎、肠病性关节炎。
本发明结合人IL-33的抗体或其抗原结合片段及其组合物在对包括人在内的动物给药时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。具体讲静脉注射的剂量是1-1800mg/天。
本发明另一方面提供了一种治疗哮喘、关节炎、特异反应性/过敏性皮炎、慢性鼻窦炎、慢性阻塞性肺病(COPD)、系统性硬化症、肝纤维化、牛皮癣、溃疡性结肠炎、克罗恩氏病、多发性硬化症、糖尿病肾脏疾病、发炎性肠道疾病、银屑病、嗜酸性食管炎、糖尿病性黄斑水肿、年龄相关性黄斑变性、干眼病、肿瘤的方法，其特征在于，给需要的对象施用上述的结合人IL-33的抗体或其抗原结合片段或药物组合物、抗体药物偶联物、或其组合。
本发明另一方面提供了一种IL-33蛋白的突变体，对应于野生型人IL-33蛋白的氨基酸序列，所述突变体包含选自下组的一个或多个位点发生突变；
(Z1)第45位赖氨酸(K45)；
(Z2)第49位缬氨酸(V49)；
(Z3)第65位天冬氨酸(D65)；
(Z4)第50位亮氨酸(L50)；
(Z5)第60位丝氨酸(S60)；
(Z6)第52位丝氨酸(S52)；
(Z7)第48位赖氨酸(K48)；
(Z8)第51位亮氨酸(L51)；
(Z9)第53位酪氨酸(Y53)；
(Z10)第55位谷氨酸(E55)。
在另一优选例中，所述野生型人IL-33蛋白的序列如SEQ ID NO.55所示。
在另一优选例中，所述突变体包含选自下组的一个或多个位点发生突变；
(Z1)第45位赖氨酸(K45)；
(Z2)第49位缬氨酸(V49)；
(Z3)第65位天冬氨酸(D65)；
(Z4)第50位亮氨酸(L50)，和/或
所述突变体包含选自下组的一个或多个位点发生突变；
(Z5)第60位丝氨酸(S60)；
(Z6)第52位丝氨酸(S52)；和/或
所述突变体包含选自下组的一个或多个位点发生突变；
(Z7)第48位赖氨酸(K48)；
(Z8)第51位亮氨酸(L51)；
(Z9)第53位酪氨酸(Y53)；
(Z10)第55位谷氨酸(E55)。
在另一优选例中，与野生型IL-33蛋白与结合人IL-33的抗体的亲和力相比，所述IL-33蛋白的突变体与结合人IL-33的抗体的亲和力下降1倍，较佳地下降5倍，更佳地下降10倍，更佳地下降25倍，最佳地下降50倍。
在另一优选例中，所述突变体还包含选自下组的一个或多个位点发生突变；
(Z11)第41位赖氨酸(K41)；
(Z12)第42位赖氨酸(K42)；
(Z13)第43位天冬氨酸(D43)；
(Z14)第44位谷氨酸(E44)；
(Z15)第46位赖氨酸(K46)；
(Z16)第47位天冬氨酸(D47)；
(Z17)第54位酪氨酸(Y54)；
(Z18)第56位丝氨酸(S56)；
(Z19)第57位谷氨酰胺(Q57)；
(Z20)第58位组氨酸(H58)；
(Z21)第59位脯氨酸(P59)；
(Z22)第61位天冬酰胺(N61)；
(Z23)第62位谷氨酸(E62)；
(Z24)第63位丝氨酸(S63)；
(Z25)第67位缬氨酸(V67)；
(Z26)第68位天冬氨酸(D68)；
(Z27)第70位赖氨酸(K70)。
在另一优选例中，所述的IL-33蛋白的突变体包含将(Z1)-(Z27)中的一个或多个氨基酸突变为丙氨酸(A)或甘氨酸(G)。
在另一优选例中，所述IL-33蛋白的突变体中的突变选自下组：
K45A、V49A、D65A、L50A、S60A、S52A、K48A、L51A、Y53A、E55A、K41A、K42A、D43A、E44A、K46A、D47A、Y54A、S56A、Q57A、H58A、P59A、N61A、E62A、S63A、V67A、D68A、K70A、或其组合；
优选下组中的一个或多个：K45A、V49A、D65A、L50A、S60A、S52A、K48A、L51A、Y53A、E55A；更优选下组中的一个或多个：K45A、V49A、D65A、L50A、S60A、S52A；最优选下组中的一个或多个：K45A、V49A、D65A、L50A。
在另一优选例中，所述IL-33蛋白的突变体选自下组：
将SEQ ID NO.:55所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-8个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有结合IL-33抗体功能的衍生的多肽。
在另一优选例中，所述IL-33蛋白的突变体的氨基酸序列与SEQ ID NO.55相比具有至少70％，优选至少75％、80％、85％、90％，更优选至少95％、96％、97％、98％、99％以上的序列相同性。
本发明另一方面提供了一种评估抗人IL-33抗体结合表位的方法，包括：
(S1)提供一抗人IL-33抗体；
(S2)与结合野生型IL-33蛋白的亲和力A2相比，检测所述抗人IL-33抗体与IL-33突变体的亲和力A1，其中所述IL-33突变体包括K45A、V49A、L50A、D65A、S60A或S52A的一种或多种位点突变，如果亲和力下降比例A1/A2≥2.5倍，较佳地≥10倍；则说明所述抗人IL-33抗体结合野生型IL-33蛋白的线性和/或空间表位包括K45、V49、L50、D65、S60A或S52A位点中的一种或多种；其中，所述抗人IL-33抗体包括：
重链互补决定区H-CDR1、H-CDR2、H-CDR3，所述H-CDR1、H-CDR2、H-CDR3的氨基酸序列分别如SEQ ID NO：18、24和25所示，和
轻链互补决定区L-CDR1、L-CDR2、L-CDR3，所述L-CDR1、L-CDR2、L-CDR3的氨基酸序列分别如SEQ ID NO：26、22和27。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：目前临床上亟待开发新型、特异、高效的IL-33强表达疾病的治疗药，从而能够改善患有此类疾病人群的生活质量，为患者提供更多、更有效的治疗方案。本发明的抗体对人IL-33具有良好的亲和力，能够阻断IL-33和其受体ST2的结合，可用于治疗多种病症，具有良好的临床应用前景。
附图说明
图1A～B：鼠源抗体对人IL-33结合活性。
图2A～B：鼠源抗体阻断ST2结合IL-33的活性。
图3A～C：鼠源抗体对IL-33诱导HUVEC细胞IL-6表达的抑制作用。
图4：鼠源抗体对IL-33诱导HUVEC细胞IL-8表达的抑制作用。
图5：鼠源抗体与食蟹猴IL-33抗原的交叉活性。
图6：脾脏称重实验评价鼠源抗体体内中和活性。
图7：嵌合抗体对人IL-33的结合活性。
图8：嵌合抗体阻断ST2结合IL-33的活性。
图9A～D：各人源化抗体对人IL-33的结合活性。
图10：人源化抗体阻断ST2结合IL-33的活性。
图11：人源化抗体对IL-33诱导HUVEC细胞IL-6表达的抑制作用。
图12：人源化抗体对IL-33诱导PBMC分泌IFNγ的抑制作用。
图13：人源化抗体对IL-33诱导NK细胞分泌IFNγ的抑制作用。
图14：人源化抗体864F3-Hu4和874F7-Hu1可有效抑制IL-33诱导KU812细胞分泌IL-5。
图15：人源化抗体864F3-Hu4和874F7-Hu1可有效抑制IL-33诱导KU812细胞分泌IL-13。
图16：脾脏称重实验评价人源化抗体体内药效活性。
图17：脾脏称重实验评价人源化抗体864F3-Hu4和874F7-Hu1的体内药效活性。
图18：人源化抗体864F3-Hu4和874F7-Hu1可有效抑制小鼠外周血IL-5分泌。
图19：人源化抗体864F3-Hu4和874F7-Hu1可有效降低小鼠外周血中由人IL-33刺激引起的嗜酸性粒细胞的增加。
图20：IL-33突变体蛋白(E44A、K45A、K46A、Q57A、H58A、P59A、S60A)对874F7-Hu1的亲和力。
图21：IL-33突变体蛋白(K48A、V49A、L51A、S52A、Y53A、E55A)对874F7-Hu1的亲和力。
图22：IL-33突变体蛋白(N61A、E62A、S63A、D65A、V67A、D68A、K70A)对874F7-Hu1的亲和力。
图23：IL-33突变体蛋白(L50A)对874F7-Hu1的亲和力。
图24：影响874F7-Hu1结合的关键氨基酸位点在IL-33结晶3D结构图中位置。
具体实施方式
本发明人经过广泛而深入地研究，经过大量筛选，获得了一系列具有优异亲 和力的抗IL-33人源化抗体。具体地，本发明的人源化抗体能够阻断IL-33和其受体ST2的结合，抑制IL-33诱导PBMC、NK细胞分泌IFNγ，并能抑制IL-33诱导嗜碱性白血病细胞KU812分泌IL-5和IL-13。本发明的人源化抗体在体内具有明显的中和活性。在小鼠动物实验中，施用本发明的人源化抗体进行单次治疗时即可有效降低小鼠外周血中由人IL-33刺激引起的嗜酸性粒细胞的增加。本发明的抗体有望用于治疗多种IL-33相关的病症。在此基础上完成了本发明。
术语
本发明中，术语“抗体(Antibody，缩写Ab)”和“免疫球蛋白G(Immunoglobulin G，缩写IgG)”是有相同结构特征的异四聚糖蛋白，其由两条相同的轻链(L)和两条相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型(isotype)的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是恒定区，重链恒定区由三个结构域CH1、CH2、以及CH3构成。每条轻链的一端有可变区(VL)，另一端有恒定区，轻链恒定区包括一个结构域CL；轻链的恒定区与重链恒定区的CH1结构域配对，轻链的可变区与重链的可变区配对。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediated cytotoxicity)等。重链恒定区包括IgG1、IgG2、IgG3、IgG4亚型；轻链恒定区包括κ(Kappa)或λ(Lambda)。抗体的重链和轻链通过重链的CH1结构域和轻链的CL结构域之间的二硫键共价连接在一起，抗体的两条重链通过铰链区之间形成的多肽间二硫键共价连接在一起。
本发明中，术语“Fab”和“Fc”是指木瓜蛋白酶可将抗体裂解为两个完全相同的Fab段和一个Fc段。Fab段由抗体的重链的VH和CH1以及轻链的VL和CL结构域组成。Fc段即可结晶片段(fragment crystallizable，Fc)，由抗体的CH2和CH3结构域组成。Fc段无抗原结合活性，是抗体与效应分子或细胞相互作用的部位。
本发明中，术语“scFv”为单链抗体(single chain antibody fragment，scFv)，由抗体重链可变区和轻链可变区通常通过15～25个氨基酸的连接短肽(linker) 连接而成。
本发明中，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于重链可变区和轻链可变区中称为互补决定区(complementarity-determining region，CDR)或超变区中的三个片段中。可变区中较保守的部分称为框架区(frame region，FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。
如本文所用，术语“框架区”(FR)指插入CDR间的氨基酸序列，即指在单一物种中不同的免疫球蛋白间相对保守的免疫球蛋白的轻链和重链可变区的那些部分。免疫球蛋白的轻链和重链各具有四个FR，分别称为FR1-L、FR2-L、FR3-L、FR4-L和FR1-H、FR2-H、FR3-H、FR4-H。相应地，轻链可变结构域可因此称作(FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L)且重链可变结构域可因此表示为(FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H)。优选地，本发明的FR是人抗体FR或其衍生物，所述人抗体FR的衍生物与天然存在的人抗体FR基本相同，即序列同一性达到85％、90％、95％、96％、97％、98％或99％。
获知CDR的氨基酸序列，本领域的技术人员可轻易确定框架区FR1-L、FR2-L、FR3-L、FR4-L和/或FR1-H、FR2-H、FR3-H、FR4-H。
如本文所用，术语“人框架区”是与天然存在的人抗体的框架区基本相同的(约85％或更多，具体地90％、95％、97％、99％或100％)框架区。
如本文所用，术语“接头”是指插入免疫球蛋白结构域中为轻链和重链的结构域提供足够的可动性以折叠成交换双重可变区免疫球蛋白的一个或多个氨基酸残基。在本发明中，优选的接头是指接头Linker1和Linker2，其中Linker1连接单链抗体(scFv)的VH和VL，而Linker2用于将scFv与另一抗体的重链进行连接。
合适的接头实例包括单甘氨酸(Gly)、或丝氨酸(Ser)残基，接头中氨基酸残基的标识和序列可随着接头中需要实现的次级结构要素的类型而变化。
在本发明中，本发明抗体还包括其保守性变异体，指与本发明双特异性抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地 至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明中，术语“抗”、“结合”、“特异性结合”是指两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。通常，抗体以小于大约10 -7M，例如小于大约10 -8M、10 -9M、10 -10M、10 -11M或更小的平衡解离常数(KD)结合该抗原。本发明中，术语“KD”是指特定抗体-抗原相互作用的平衡解离常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。例如，使用表面等离子体共振术(Surface Plasmon Resonance，缩写SPR)在BIACORE仪中测定抗体与抗原的结合亲和力或使用ELISA测定抗体与抗原结合的相对亲和力。
本发明中，术语“表位”是指与抗体特异性结合的多肽决定簇。本发明的表位 是抗原中被抗体结合的区域。
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
药物组合物和应用
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射(如腹膜内)、颅内注射、或腔内注射。本发明中，术语“药物组合物”是指本发明的双特异性抗体可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的双特异性抗体的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。本发明的药物组合物含有安全有效量(如0.001-99wt％，较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的双特异性抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的双特异性抗体还可与 其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的双特异性抗体或其免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
抗体-药物偶联物(ADC)
本发明还提供了基于本发明抗体的抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基 的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier) 中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2021120785-appb-000001
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1到8的值。
以下实施例是对本发明进行进一步的说明，不应理解为是对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法.这样的方法对本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais,T.(1989)Molecular Cloning:A Laboratory Manual,2 nd edition,Cold spring Harbor Laboratory Press.
以下实施例中使用的实验材料和来源以及实验试剂的配制方法具体说明如下。
实验材料及试剂：
Balb/c小鼠：购自上海灵畅生物科技有限公司。
PBMC：购自澳赛尔斯生物技术上海有限公司，货号PB004-C。
人IL-33-his：将IL-33序列的Ser112-Thr270(NCBI登录号为NP_254274.1)克隆至E.coli表达载体进行表达，并在IL-33的C-末端添加6×His标签以便于通过Ni +亲和层析柱进行纯化，其氨基酸序列如SEQ ID NO：53所示。
食蟹猴IL-33-his：购自义翘神州，货号90912-CNAE。
IL-33-his-biotin蛋白：购自Thermo Fisher，货号20217，按照EZ-Link NHS-Biotin Reagent试剂说明书对IL-33-his蛋白进行biotin化。
Rat-Anti-human IL-6：购自BD Pharmingen，货号554543。
biotin Rat Anti Human IL-6：购自BD Pharmingen，货号554546。
Mouse-Anti-human IFNγ：购自BD Biosciences，货号551221。
biotin mouse-anti-human IFNγ：购自BD Biosciences，货号554550。
羊抗小鼠IgG二抗：购自Millipore，货号AP181P。
HRP-anti human IgG Fc二抗：购自Sigma，货号A0170。
HRP标记的streptavidin二抗：购自BD Biosciences，货号554066。
IL-6试剂盒：Invitrogen，货号88-7066-77。
IL-8试剂盒：Invitrogen，货号BMS204-3TEN
IL-12：购自Sino Biological，货号CT011-H08H。
ST2-Fc蛋白：将人的ST2序列(NCBI登录号为NP_003847.2)与人IgG1的Fc区序列进行融合克隆至真核表达载体PTT5中，通过转染HEK-293F细胞进行融合表达，随后收集表达上清通过ProteinA亲和层析柱进行纯化。ST2-Fc氨基酸序列如SEQ ID NO：54所示。
TMB：购自BD公司，货号555214。
1％的Glutmine(谷氨酰胺)、1％Sodium pyruvate(丙酮酸钠)、1％MEM-NEAA(最小基本培养基-非必需氨基酸溶液)、1％Penicillin-streptomycin(青霉素-链霉素)、β-巯基乙醇、20％FBS(胎牛血清)：均购自Gibco公司。
SA蛋白：Streptavidin，购自Sigma，货号85878-1MG。
SFM培养基：购自life technologies公司，货号12045-076。
RPMI1640完全培养基：购自Gibco。
实验仪器：
电融合仪：购自BTX公司。
酶标仪：购自Molecular Devices，型号SpectraMax 190。
本发明的抗体序列如下表所示：
Figure PCTCN2021120785-appb-000002
Figure PCTCN2021120785-appb-000003
Figure PCTCN2021120785-appb-000004
实施例1 抗原免疫动物以及杂交瘤的制备和筛选
步骤1：抗原免疫小鼠
用原核重组表达的人IL-33-his蛋白常规腹腔免疫Balb/c小鼠。第一天，可溶性人IL-33-his蛋白与弗氏完全佐剂乳化或水溶性佐剂(quick antibody)充分混匀后，对Balb/c小鼠进行腹腔注射(人IL-33-his 50μg/鼠)，第十四天，可溶性人IL-33-his蛋白与弗氏不完全佐剂乳化或水溶性佐剂(quick antibody)充分混匀后，对Balb/c小鼠进行腹腔加强免疫(人IL-33-his 50μg/鼠)，在第三十六天，用可溶性人IL-33-his蛋白同上次一样加强免疫动物(人IL-33-his 50μg/小鼠)，三周以后腹腔内注射人IL-33-his激发，3～4天后，取小鼠脾脏进行融合实验。
步骤2：杂交瘤的制备和筛选
在小鼠末次冲击免疫后3～4天，使用常规的杂交瘤技术方案，将小鼠脾细胞与小鼠骨髓瘤细胞SP2/0通过电融合仪进行电融合。融合后的细胞在完全培养基中悬浮均匀，完全培养基即将RPMI1640和DMEM F12培养基1：1混匀后加入1％的Glutmine(谷氨酰胺)，1％Sodium pyruvate(丙酮酸钠)，1％MEM-NEAA (最小基本培养基-非必需氨基酸溶液)，1％Penicillin-streptomycin(青霉素-链霉素)，50μM的β-巯基乙醇及20％FBS(胎牛血清)组成的培养基；按10 5个细胞/100μl/孔，分入共36块96孔培养板中培养过夜，次日，每孔加入100μl孔含有2×HAT的完全培养基，使96孔板内培养液为200μl/孔(含1×HAT)。在7～12天后，收获上清液，通过间接酶联免疫吸附测定法(ELISA)筛选人IL-33-his结合活性阳性的杂交瘤孔。
其中，间接酶联免疫吸附测定法筛选人IL-33-his结合活性阳性的杂交瘤孔的方法如下：将重组人IL-33-his蛋白以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释至1μg/ml，100μl/孔加入酶标板，4℃包被过夜。PBST洗板3次，加入200μl/孔封闭液(2％BSA-PBS)，37℃放置1h后PBST洗板1次待用。将收取的杂交瘤上清液依次加入封闭后的酶标板，100μl/孔，37℃放置1h。PBST洗板3次，加入HRP标记的羊抗小鼠IgG二抗，37℃放置30min；PBST洗板5次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5min；每孔加入50μl 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析待测抗体与靶抗原人IL-33-his结合能力。
在含血清完全培养基中扩增筛选获得的10株杂交瘤细胞株，离心换液至无血清培养液SFM培养基，使细胞密度为1～2×10 7/ml，在5％CO 2，37℃条件下培养2周，离心获取培养上清，通过Protein G亲和层析进行纯化，获得10株鼠源抗人IL-33单克隆抗体。分别命名为874F7、871G1、864F3、887B9、858D5、868H10、604A8、604A12、646F8、651H2。
实施例2 ELISA法测定鼠源抗体对人IL-33的结合活性
间接酶联免疫吸附测定法测定鼠源抗体对人IL-33的结合能力。具体方法如下：预先包被生物素-亲和素(SA)以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释至2μg/ml包板4℃，过夜；再用5％的脱脂奶粉封闭，37℃，2小时。储存于-80℃待用。将生物素化的重组人IL-33-his蛋白以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释至0.5μg/ml，100μl/孔加入预包被的SA的板子中，37℃，0.5小时。PBST洗板3次，将以1％BSA梯度稀释的待测抗体依次加入封闭后的酶标板，100μl/孔，37℃放置1h。PBST洗板3次，加入HRP标记的羊抗小鼠IgG二抗，37℃放置30min；PBST洗板3次后，在吸水纸上尽量拍干残留 液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5min；每孔加入50μl 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析待测抗体与靶抗原人IL-33-his的结合能力。
结果如图1A、1B，所示抗体都有很好的结合活性。874F7、871G1、864F3、887B9、858D5、868H10抗体EC 50分别为0.11nM、0.14nM、0.27nM、0.07nM、0.18nM、0.36nM。604A8、604A12、646F8、651H2抗体EC 50分别为0.11nM、0.16nM、0.10nM、0.13nM。
实施例3 鼠源抗体阻断ST2结合IL-33的活性测定
ST2是目前被发现的IL-33唯一的受体，IL-33作为细胞因子，依赖ST2信号通路发挥其生理功能，抗体的阻断活性可以反映其中和活性。具体方法如下：以2μg/ml ST2PBS溶液包板，4℃过夜；PBST洗涤3次，再用5％的脱脂奶粉封闭，37℃，2小时；PBST洗涤3次待用；1％BSA稀释的20ng/ml IL-33-his-biotin抗原与用1％BSA梯度稀释待测抗体1:1等体积混合，37℃孵育小时；将此混合液加入到预先包被的ST2板中，37℃孵育1小时；PBST洗涤3次；按1:8000加入HRP标记的streptavidin二抗，37℃孵育0.5小时；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5min；每孔加入50μl 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值。
结果见图2A、2B，图2A所示的抗体874F7、871G1、864F3、887B9、858D5、868H10都有很好的阻断活性，IC 50分别为0.90nM、0.74nM、0.23nM、0.25nM、0.16nM、0.27nM。图2B所示抗体604A8、604A12、646F8、651H2没有或有很弱的阻断活性。
实施例4 鼠源抗体对IL-33诱导HUVEC细胞IL-6表达的抑制作用
采用HUVEC细胞测定抗IL-33抗体的生物学活性。具体方法如下：在T75培养瓶中培养HUVEC细胞，把HUVEC细胞传至第五代细胞，将T75培养瓶中的HUVEC细胞用1×PBS清洗，再用1ml胰酶消化10min，待细胞脱落，用10ml培养基终止消化，计数，铺板，100μl/孔，6000个/孔。剩余于T75培养瓶中继 续培养冻存。IL-33-his以培养基稀释至20ng/ml，待测抗体从40μg/ml，4倍稀释8个梯度，空白做4个孔，稀释后抗原和抗体取各取50μl加入细胞板中，混匀。即IL-33-his的终浓度为5ng/ml。混合后37℃孵育12h～18h。取上清测定IL-6的浓度。测定方法详见IL-6试剂盒说明书。按照以下公式计算IL-6抑制率并通过GraphPad Prism 6软件进行拟合分析：
抑制率＝(ODAgIL-33-OD给药)/(ODAgIL-33-OD空白)×100％。
结果如图3A、3B、3C，874F7、871G1、864F3、887B9、858D5、868H10、604A8、604A12、651H2抑制活性的IC 50分别为0.65nM、1.66nM、1.52nM、4.53nM、12.25nM、9.35nM、0.08nM、0.13nM、0.04nM。另外，抗体604A8、604A12、651H2的最大抑制率较差。
实施例5 鼠源抗体对IL-33诱导HUVEC细胞IL-8表达的抑制作用
采用HUVEC细胞测定抗IL-33抗体的生物学活性。具体方法如下：在T75培养瓶中培养HUVEC细胞，把HUVEC细胞传至第五代细胞，将T75培养瓶中的HUVEC细胞用1×PBS清洗，再用1ml胰酶消化10min，待细胞脱落，用10ml培养基终止消化，计数，铺板，100μl/孔，6000个/孔。剩余于T75培养瓶中继续培养冻存。IL-33-his以培养基稀释至20ng/ml，待测抗体从40μg/ml，4倍稀释8个梯度，空白做4个孔，稀释后抗原和抗体取各取50μl加入细胞板中，混匀。即IL-33-his的终浓度为5ng/ml。混合后37℃孵育12h～18h。取上清测定IL-8的浓度。测定方法详见IL-8试剂盒说明书。按照以下公式计算IL-8抑制率并通过GraphPad Prism 6软件进行拟合分析：
抑制率＝(ODAgIL-33-OD给药)/(ODAgIL-33-OD空白)×100％。
结果见图4，抗体858D5、868H10活性很弱，抗体874F7、871G1、864F3、887B9的IC 50分别为0.35nM、0.16nM、1.46nM、8.90nM。
实施例6 鼠源抗体与食蟹猴IL-33抗原的交叉活性测定
间接酶联免疫吸附测定法测定鼠源抗体对食蟹猴IL-33的结合能力。具体方法方法如下：预先包被生物素-亲和素(SA)以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释至2μg/ml包板4℃，过夜；再用5％的脱脂奶粉封闭，37℃， 2小时。储存于-80℃待用。将生物素化的重组食蟹猴IL-33-his蛋白以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释至1μg/ml，100μl/孔加入预包被的SA的板子中，37℃，0.5小时。PBST洗板3次，将以1％BSA梯度稀释的待测抗体依次加入封闭后的酶标板，100μl/孔，37℃放置1h。PBST洗板3次，加入HRP标记的羊抗小鼠IgG二抗，37℃放置30min；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5min；每孔加入50μl 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析待测抗体与靶抗原食蟹猴IL-33-his的结合能力。
结果见图5，所示抗体都能很好的结合食蟹猴IL-33蛋白。抗体874F7、871G1、864F3、887B9、858D5、868H10、604A8、604A12、646F8、651H2的EC 50分别为0.03nM、0.04nM、0.06nM、0.05nM、0.02nM、0.05nM、0.06nM、0.06nM、0.03nM、0.06nM。
实施例7 鼠源抗体体内中和活性评价
动物静脉给抗原IL-33会引起体内炎症反应改变，脾脏细胞增殖进而引起脾脏重量增加，此方法可以用来评价抗体药物在体内中和IL-33的活性。具体方法如下：雌性BALB/C小鼠，体重18-20g，随机分成6组，每组10只动物。第一组，正常对照组；第二组，IL-33-his抗原攻击组，分组后第二天开始每天腹腔注射0.4ug/只IL-33抗原，连续给6天；第三组，864F3治疗组，分组当天，腹腔注射864F3抗体，5mg/kg，分组后第二天开始每天腹腔注射0.4ug/只IL-33抗原，连续给6天；第四组，871G1治疗组，分组当天，腹腔注射871G1抗体，5mg/kg，分组后第二天开始每天腹腔注射0.4ug/只IL-33-his抗原，连续给6天；第五组，874F7治疗组，分组当天，腹腔注射874F7抗体，5mg/kg，分组后第二天开始每天腹腔注射0.4ug/只IL-33抗原，连续给6天。分组后第7天，动物称重，安乐死动物取脾脏称重。
实验结果见图6，PBS对照组脾脏重量平均值为86.9±9.4mg，而给抗原Ag-IL-33组增重明显为195.7±26.9mg。864F3、871G1、874F7抗体给药组脾脏重量平均值分别为106.3±24.5mg、77.7±17.5mg、89.9±14.7mg，都具有明显的中和活性。
实施例8 嵌合抗体的制备
本实施例通过分子生物学的相关方法获取杂交瘤864F3、874F7和871G1的重链可变区和轻链可变区，并进一步构建嵌合抗体。
通过Trizol提取864F3、874F7和871G1三个杂交瘤细胞的RNA并进行mRNA反转录获取cDNA，随后以cDNA为模板，分别用鼠源抗体的重链和轻链简并引物(《Antibody Engineering》Volume 1，Edited by Roland Kontermann and Stefan Dübel，组合引物的序列来自第323页)进行PCR，对所获得的PCR产物进行测序并通过kabat数据库分析，确定所获得的序列为鼠源抗体的可变区序列。
相关序列信息如下：
864F3具有两个重链可变区基因序列，全长均为363bp，各自编码121个氨基酸残基，核苷酸序列分别如SEQ ID NO：1和SEQ ID NO：3所示，氨基酸序列分别如SEQ ID NO：2和SEQ ID NO：4所示；864F3轻链可变区基因序列全长321bp，编码107个氨基酸残基，核苷酸序列如SEQ ID NO：5所示，氨基酸序列如SEQ ID NO：6所示。
874F7重链可变区基因序列全长为363bp，编码121个氨基酸残基，核苷酸序列如SEQ ID NO：7所示，氨基酸序列如SEQ ID NO：8所示；874F7轻链可变区基因序列全长318bp，编码106个氨基酸残基，核苷酸序列如SEQ ID NO：9所示，氨基酸序列如SEQ ID NO：10所示。
871G1重链可变区基因序列全长为363bp，编码121个氨基酸残基，核苷酸序列如SEQ ID NO：3所示，氨基酸序列如SEQ ID NO：4所示；871G1轻链可变区基因序列全长318bp，编码106个氨基酸残基，核苷酸序列如SEQ ID NO：11所示，氨基酸序列如SEQ ID NO：12所示。
三种优选的鼠源抗IL-33抗体的3个H-CDR和3个L-CDR的同源性较高，尤其是均具有相同的L-CDR2(SEQ ID No:22)。
对所得的各杂交瘤重链可变区序列与人的IgG4恒定区(包含S228P突变)(氨基酸序列如SEQ ID NO：13所示)拼接，轻链可变区序列与人的kappa链恒定区(氨基酸序列如SEQ ID NO：14所示)拼接，分别构建各嵌合抗体的重链和轻链至pcDNA3.4表达载体，转染HEK-293F细胞，通过Protein A纯化获得各嵌合抗体，通过SDS-PAGE电泳及SEC-HPLC确定所表达各抗体分子量在 150kD左右，抗体纯度>95％，定量，分装，冻存于-80℃备用。
实施例9 ELISA法测定各嵌合抗体对人IL-33的结合活性
将SA蛋白稀释至2μg/mL，包被ELISA板，100μL/孔，4℃包被过夜，PBST(PBS含0.05％Tween20)洗涤3次，用PBS配制2％BSA，200μL/孔，于室温封闭2h，PBST洗涤2次后，用PBST配制的1％BSA将IL-33-his-biotin蛋白稀释至1μg/mL，按照100μL/孔加至ELISA板中，室温孵育1h，用PBST配制的1％BSA按3倍梯度分别稀释各单克隆抗体，最高浓度为10μg/mL，稀释12个梯度，加入ELISA孔，100μL/孔，室温孵育1h，每个样品平行做2个复孔，PBST洗涤3次，用PBST配制的1％BSA按照适当比例稀释HRP-anti human IgG Fc二抗，加入ELISA孔，100μL/孔，室温孵育1h，PBST洗涤3次后添加TMB显色液，100μL/孔，显色至预期颜色，用2M H 2SO 4终止显色反应，70μL/孔，使各反应液振荡均匀并于酶标仪测定OD450nm，分析数据，计算EC 50。
实验结果如图7所示，嵌合抗体864F3-ch2、864F3-ch1、871G1-ch、874F7-ch的EC 50分别为0.173nM、0.127nM、0.178nM、0144nM，表明各嵌合抗体对IL-33均具有良好的亲和力。
实施例10 嵌合抗体阻断ST2结合IL-33的活性测定
用ELISA包被液将ST2-Fc蛋白稀释至1μg/mL，包被ELISA板，100μL/孔，置于湿盒中，4℃，包被16h，PBST洗涤ELISA板3次，然后用PBS配制的2％BSA，200μL/孔，于室温封闭2h，PBST洗涤1次，拍干，除去多余的封闭液，用1％BSA PBST按3倍梯度分别稀释各单克隆抗体，最高浓度为40μg/mL，稀释11个梯度，将稀释好的抗体与10ng/ml IL-33-his-biotin蛋白1:1混合，混合均匀后加入ELISA孔，100μL/孔，室温孵育1h，每个浓度平行做2个复孔(抗体最高浓度终浓度为20μg/mL，IL-33-his-biotin终浓度为5ng/mL)，洗除未结合的或非特异性结合的一抗，按照抗体说明书要求，用抗体稀释液HRP标记的streptavidin二抗稀释至合适浓度，加入ELISA板，100μL/孔，室温孵育1h，用PBST洗涤5次，并将ELISA板于吸水纸上拍干，除去多余的液体，加入TMB显色液，100μL/孔，显色至合适深浅，加入2M H 2SO 4，70μL/孔，以终 止显色，并于多功能酶标仪中在450nm波长处测定其吸光度，分析数据。
实验结果如图8所示，嵌合抗体874F7-ch、871G1-ch、864F3-ch1、864F3-ch2的IC 50分别为0.634nM、0.853nM、45.245nM、0.580nM，表明各嵌合抗体为IL-33阻断型抗体，均能有效阻断IL-33与其受体ST2的结合，并且874F7-ch、871G1-ch和864F3-ch2较优。
实施例11 人源化抗体的制备
对各候选鼠源抗体轻链可变区和重链可变区的氨基酸序列进行分析，依据Kabat规则确定鼠源抗体的3个抗原互补决定区(CDR)和4个框架区(FR)。其中，864F3重链互补决定区的氨基酸序列为
HCDR1：NYGVH(SEQ ID NO：15)、
HCDR2：VIRAGGSSDYNSALMS(SEQ ID NO：16)、
HCDR3：DHYFSNSYGGSPY(SEQ ID NO：17)或
HCDR1：KYGVH(SEQ ID NO：18)、
HCDR2：VLRAGGTISYNSALMS(SEQ ID NO：19)、
HCDR3：DHYYYSSFGGFAS(SEQ ID NO：20)，
轻链互补决定区的氨基酸序列为
LCDR1：LASQTIATWLA(SEQ ID NO：21)、
LCDR2：AATRLAD(SEQ ID NO：22)和
LCDR3：QQLYNTPYT(SEQ ID NO：23)。
874F7重链互补决定区的氨基酸序列为
HCDR1：KYGVH(SEQ ID NO：18)、
HCDR2：VLRAGGSTGYNSALMS(SEQ ID NO：24)、
HCDR3：DHYYYSSYGGFVY(SEQ ID NO：25)，
轻链互补决定区的氨基酸序列为
LCDR1：LASQTIGAWLA(SEQ ID NO：26)、
LCDR2：AATRLAD(SEQ ID NO：22)和
LCDR3：QQLDSSPYT(SEQ ID NO：27)。
871G1重链互补决定区的氨基酸序列为
HCDR1：KYGVH(SEQ ID NO：18)、
HCDR2：VLRAGGTISYNSALMS(SEQ ID NO：19)、
HCDR3：DHYYYSSFGGFAS(SEQ ID NO：20)，
轻链互补决定区的氨基酸序列为
LCDR1：LASQTIGAWLA(SEQ ID NO：26)、
LCDR2：AATRLAD(SEQ ID NO：22)和
LCDR3：QQLNSTPYT(SEQ ID NO：28)。
鼠源抗体的CDR序列如下表1a所示：
Figure PCTCN2021120785-appb-000005
在Germline数据库中选取与上述各鼠源抗体非FR区匹配最好的人源化模板。然后将鼠源抗体的CDR区移植到所选择的人源化模板上，替换人源模板的CDR区，重链可变区再与人IgG4恒定区(包含S228P突变)重组，轻链可变区与人的kappa链恒定区重组，同时以该抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对各抗体的VL和VH的构象有重要影响的残基进行回复突变，最终获得多个人源化抗体，各人源化抗体和嵌合抗体对应的 重链、轻链可变区及序列如表1b所示。分别构建各人源化抗体的重链和轻链至pcDNA3.4表达载体，转染HEK-293F细胞，通过ProteinA纯化获得各人源化抗体，并通过SDS-PAGE电泳及SEC-HPLC确定各抗体分子量大小正确及纯度>95％。
表1b：各人源化抗体可变区序列表
Figure PCTCN2021120785-appb-000006
Figure PCTCN2021120785-appb-000007
实施例12 ELISA法测定各人源化抗体对人IL-33的结合活性
通过ELISA法测定上述各人源化抗体对人IL-33的结合亲和力，相关实验方法参照实施例9。
实验结果如图9A～9D所示，嵌合抗体864F3-ch2和人源化抗体864F3-HuG、864F3-Hu1、864F3-Hu2的EC 50分别为0.169nM、0.760nM、0.249nM、0.181nM，与嵌合抗体864F3-ch2相比，人源化抗体864F3-HuG、864F3-Hu1、864F3-Hu2对人IL-33的亲和力损失较多，嵌合抗体864F3-ch2和864F3-Hu3、864F3-Hu4、864F3-Hu5的EC 50分别为0.124nM、0.138nM、0.135nM、0.146nM，与嵌合抗体864F3-ch2相比，864F3-Hu3、864F3-Hu4和864F3-Hu5对人IL-33的亲和力几乎不变。
嵌合抗体874F7-ch和人源化抗体874F7-Hμg、874F7-Hu1、874F7-Hu2的EC 50分别为0.177nM、0.170nM、0.129nM、0.136nM。与嵌合抗体874F7-ch相比，人源化抗体874F7-Hu1和874F7-Hu2对人IL-33的亲和力未发生损失。
嵌合抗体871G1-ch和人源化抗体871G1-HuG、871G1-Hu1、871G1-Hu2的EC 50分别为与0.255nM、0.187nM、0.191nM、0.176nM。与嵌合抗体871G1-ch相比，人源化抗体871G1-Hu1、871G1-Hu2和871G1-HuG对人IL-33的亲和力均未发生损失。
实施例13 人源化抗体阻断ST2结合IL-33的活性测定
通过ELISA法测定上述各人源化抗体对IL-33和其受体ST2结合的阻断作用，相关实验方法参照实施例10。
实验结果如图10所示，人源化抗体864F3-Hu4、864F3-Hu5、871G1-Hu1、871G1-Hu2、874F7-Hu1、874F7-Hu2对IL-33与ST2蛋白结合阻断作用的IC 50值分别为0.512nM、0.473nM、0.404nM、0.361nM、0.451nM、0.350nM。表明各人源化抗体均保留了对IL-33与ST2结合的阻断作用。
实施例14 人源化单克隆抗体对IL-33的结合动力学测定
利用共价偶联有Protein A/G的芯片(购自GE Healthcare，货号BR-1005-30)补获各待测抗体，相关运行参数如下：抗体浓度为2μg/mL，接触时间75s，流速10μL/min，再生接触时间为30s。利用HBS-EP pH7.4缓冲液(购自GE Healthcare，货号BR-1006-69)稀释IL-33-his抗原，最高浓度为50nM，按照2倍稀释至0.39nM，设置复孔及0浓度点，采用6M盐酸胍溶液作为再生缓冲液，在Biacore 8K上按照如下参数进样，结合时间180s，解离时间900s，流速30μL/min，再生接触时间为30s。运行完成后，利用Biacore 8K Evaluation Software按照“1：1binding kinetics model”对数据进行分析，得出各抗体对IL-33的结合动力学参数。
结果如表2，各人源化抗体对IL-33结合常数(ka)、解离常数(kd)以及平衡解离常数(KD)均处于同一水平。
表2：各人源化抗体对IL-33的结合动力学参数
样品 ka(1/Ms) kd(1/s) KD(M)
864F3-Hu4 3.23E+05 4.47E-04 1.38E-09
864F3-Hu5 3.23E+05 4.39E-04 1.36E-09
874F7-Hu1 2.02E+05 5.70E-04 2.82E-09
874F7-Hu2 1.94E+05 5.76E-04 2.97E-09
871G1-Hu1 2.95E+05 5.04E-04 1.71E-09
871G1-Hu2 3.00E+05 5.27E-04 1.76E-09
实施例15 人源化抗体对IL-33诱导HUVEC细胞IL-6表达的抑制作用
本实施例通过测定各人源化抗体对IL-33-his蛋白诱导HUVEC细胞表达IL-6的抑制效果以评价各人源化抗体的活性。具体实验步骤如下：将T75培养瓶中的HUVEC细胞消化计数后铺板于完全培养基中，100μL/孔，6000细胞/孔， 于37℃培养箱培养，2h后，待细胞贴壁后开始给药；用完全培养基稀释待测抗体，最高浓度为200nM，5倍稀释9个梯度，设置阳性空白对照及阴性空白对照；稀释后抗体与20ng/mL IL-33-his按1:1体积混合(抗体加入细胞后最高浓度终浓度为50nM，IL-33-his终浓度为5ng/mL)。室温孵育30min；取100μL抗体/IL-33-his混合液加入细胞液中轻轻混匀，37℃CO 2培养箱作用20h后离心收集上清保存于-80℃，以进行IL-6测定。
用ELISA包被液稀释Rat-Anti-human IL-6蛋白至2.5μg/mL，包被ELISA板，100μL/孔，置于湿盒中，4℃，包被16h；用PBST洗涤ELISA板三次，去除未结合蛋白，并将ELISA板于吸水纸上拍干，除去多余的液体，然后用PBS配制的2％BSA，200μL/孔，于室温封闭1-2h；用PBS配制的1％BSA按照3倍梯度稀释IL-6标准品，最高浓度为30ng/mL。混合均匀后加入ELISA孔，100μL/孔，加入100μL/孔上述细胞上清液到ELISA孔中，室温孵育1h，标准曲线每个样品平行做2个复孔；洗除未结合的或非特异性结合的一抗，用抗体稀释液将biotin Rat Anti Human IL-6按照1：1000稀释，混合均匀后加入ELISA孔，100μL/孔，室温孵育1h；洗除未结合的或非特异性结合的抗体,将HRP标记的streptavidin二抗稀释至合适浓度，加入ELISA板，100μL/孔，室温孵育1h；用PBST洗涤五次，并将ELISA板于吸水纸上拍干，除去多余的液体，加入TMB显色液，显色至合适深浅，加入2M H 2SO 4，50μL/孔，以终止显色，并于多功能酶标仪中在450nm波长处测定其吸光度，分析数据。
实验结果如图11所示，人源化抗体864F3-Hu4、864F3-Hu5、871G1-Hu1、871G1-Hu2、874F7-Hu1、874F7-Hu2对IL-33诱导HUVEC细胞的IL-6表达抑制作用IC 50值分别为0.073nM、0.147nM、0.210nM、0.175nM、0.051nM、0.050nM。表明各人源化抗体均能有效的抑制IL-33-his蛋白诱导HUVEC细胞的IL-6分泌，尤以874F7-Hu1和874F7-Hu2效果相对较优。
实施例16 人源化抗体对IL-33诱导PBMC分泌IFNγ的抑制作用
本实施例通过测定各人源化抗体对IL-33-his蛋白诱导PBMC分泌IFNγ的抑制作用以评价各人源化抗体的活性。具体实验步骤如下：将新鲜PBMC离心计数，PBS洗涤一次，用RPMI1640完全培养基稀释至4×10 6细胞/Ml；向96 孔U型细胞培养板中加入PBMC细胞，每孔50μL(2×10 5细胞/孔)，置于37℃培养箱恢复；用RPMI1640完全培养基稀释抗体，最高浓度为200nM(终浓度为50nM)，按照3倍梯度稀释，稀释后每孔加入等体积40ng/mL IL-33-his(终浓度为10ng/mL)，37℃孵育30min；向上述PBMC细胞培养板中加入50μL 40ng/mL完全培养基稀释的IL-12，随后后加入100μL抗体与IL-33-his混合液，混匀后，置于37℃细胞培养箱中培养24h；将细胞培养板置于500g离心5min，各孔吸取180μL上清，供检测。
用ELISA包被液将Mouse-Anti-human IFNγ抗体(购自BD Biosciences，货号551221)稀释至1μg/mL，包被ELISA板，100μL/孔，置于湿盒中，4℃，包被16h；用PBST洗涤ELISA板三次，并将ELISA板于吸水纸上拍干，除去多余的液体，然后用PBS配制的2％BSA，200μL/孔，于室温封闭1-2h；用PBST洗涤一次，去除多余的封闭液，并将ELISA板拍干，除去多余的液体；稀释IFNγ标准品，最高浓度为250ng/mL，1/2稀释12个梯度，加入ELISA孔，100μL/孔，每个样品平行做2个复孔；向ELISA板中加入100μL上述待测细胞上清，室温孵育1h；用PBST洗去三次，加入用1％BSA PBST 1:1000稀释的biotin mouse-anti-human IFNγ(购自BD，货号554550)，100μL/孔，室温孵育1h；洗除未结合的或非特异性结合的抗体，按照抗体说明书要求，用1％BSA PBST将HRP标记的Streptavidin(购自BD，货号554066)稀释至合适浓度，加入ELISA板，100μL/孔，室温孵育1h；用PBST洗涤五次，并将ELISA板于吸水纸上拍干，除去多余的液体，加入TMB显色液，100μL/孔，显色至合适深浅，加入2M H 2SO 4，50μL/孔，以终止显色，并于多功能酶标仪中在450nm波长处测定其吸光度，分析数据。
实验结果如图12所示，人源化抗体864F3-Hu4、864F3-Hu5、871G1-Hu1、871G1-Hu2、874F7-Hu1、874F7-Hu2对IL-33诱导PBMC分泌IFNγ的抑制作用IC 50值分别为1.224nM、0.839nM、1.395nM、1.061nM、0.850nM、1.736nM。表明各人源化抗体均能有效的抑制IL-33-his蛋白诱导人PBMC分泌IFNγ，其中874F7-Hu2的活性相对较差。
实施例17 人源化抗体对IL-33诱导NK细胞分泌IFNγ的抑制作用
将新鲜PBMC离心计数后按照Nk Cell Isolation Kit human(购自Miltenyi，货号130-092-657)说明书分离出NK细胞。用PBS洗涤NK细胞两次后计数，并用1640+10％FBS+1％Glutamax培养基稀释至0.8×10 6细胞/mL，并按照每孔50μL细胞悬液铺制96孔细胞培养板。用1640+10％FBS+1％Glutamax培养基稀释各抗体，最终最高作用浓度为100nM，1/4梯度稀释。稀释后每孔加入终浓度为10ng/mL的IL-33-his于37℃细胞培养箱孵育30min。随后向上含有NK细胞的96孔细胞培养板中加入完全培养基稀释的终浓度为2ng/mL的IL-12，最后加入上述100μL抗体与IL-33的混合液，于37℃细胞培养箱孵育24h后，收集细胞培养上清测定IFNγ的分泌水平。
IFNγ的检测方法参照实施例16。实验结果如图13所示，人源化抗体864F3-Hu4、864F3-Hu5、871G1-Hu1、871G1-Hu2、874F7-Hu1、874F7-Hu2对IL-33诱导NK细胞分泌IFNγ有抑制作用，IC 50值分别为0.904nM、1.021nM、0.851nM、0.900nM、0.742nM、1.559nM。
实施例18 人源化抗体对IL-33诱导KU812细胞分泌IL-5和IL-13的抑制作用收集处于对数生长期的嗜碱性白血病细胞KU812，离心，计数，用含20％FBS的IMDM培养基(完全培养基，购自Gibco，货号11965-092)重悬，按照20000个细胞每孔，铺制96孔细胞培养板。用完全培养基配制各待测抗体，按照3倍梯度稀释作为给药组，各组均连续稀释9梯度，抗体最高工作浓度为200μg/mL，同时以完全培养基配制IL-33-his，工作浓度为700ng/ml，按照1：1混匀后，加入上述96孔细胞培养板，每孔终体积为200μL，设置未给药组作为阴性对照，设置只添加IL-33为单药对照组，每个浓度平行做2个复孔，于37℃细胞培养箱继续培养48h，收集上清进行IL-5和IL-13分泌量的测定。
用ELISA包被液将anti-human IL-5(购自BD，货号554488)及anti-human IL-13(购自BD，货号554570)抗体分别稀释至5ug/ml，包被ELISA板，100μL/孔，置于湿盒中，4℃，包被16h。用PBST洗涤ELISA板三次，去除未结合抗原，并将ELISA板于吸水纸上拍干，除去多余的液体，然后用PBS配制的2％BSA，200μL/孔，于室温封闭1h。用PBST洗涤一次，洗除多余的封闭液，并将ELISA板拍干，除去多余的液体，用PBST配制的1％BSA按3倍梯度分别 稀释hu-IL-5标准品，最高浓度为50ng/mL，稀释12个梯度，加入ELISA孔，100μL/孔，室温孵育1h，每个样品平行做2个复孔。加入100μL/孔收集的细胞上清。用PBST配制的1％BSA按3倍梯度分别稀释hu-IL-13标准品，最高浓度为10ng/mL，稀释12个梯度，加入ELISA孔，100μL/孔，室温孵育2h，每个样品平行做2个复孔。加入50μL/孔收集的细胞上清。洗除未结合的或非特异性结合的一抗，按照抗体说明书要求，用抗体稀释液将Biotin-anti-human-IL-5(购自BD，货号554491)及Biotin-anti-human-IL-13(购自BD，货号555054)二抗稀释至稀释至合适浓度，加入ELISA板，100μL/孔，室温孵育2h。用PBST洗涤三次，并将ELISA板于吸水纸上拍干，除去多余的液体，用抗体稀释液将HRP标记的Streptavidin(购自BD，货号554066)稀释至合适浓度，加入ELISA板，100μL/孔，室温孵育0.5h。用PBST洗涤三次，并将ELISA板于吸水纸上拍干，除去多余的液体，加入TMB显色液，100μL/孔，显色至合适深浅，加入2M H 2SO 4，50μL/孔，以终止显色，并于多功能酶标仪中在450nm波长处测定其吸光度，分析数据。
实验结果如图14所示，人源化抗体864F3-Hu4和874F7-Hu1可有效抑制IL-33诱导KU812细胞分泌IL-5，其IC 50分别为27.605nM和7.828nM。
如图15所示，人源化抗体864F3-Hu4和874F7-Hu1亦可有效抑制IL-33诱导KU812细胞分泌IL-13，其IC 50分别为14.745nM和5.219nM。
实施例19 人源化抗体对不同种属IL-33蛋白的交叉反应性
实验方法参照实施例14，其中抗原分别用食蟹猴IL-33蛋白(购自Sino biological，货号90912-CNAE)和小鼠IL-33蛋白(购自R&D，3626-ML-010/CF)，同时设置人IL-33蛋白作为对照。实验结果如表3所示，864F3-Hu4和874F7-Hu1均与食蟹猴IL-33蛋白有交叉反应，并且与各抗体和人的IL-33蛋白结合特征相一致。此外，864F3-Hu4和874F7-Hu1与小鼠IL-33蛋白也有一定的交叉反应，但其亲和力要明显低于各抗体与人IL-33蛋白的结合。
表3：人源化抗体对食蟹猴和小鼠IL-33蛋白的交叉反应性
Figure PCTCN2021120785-appb-000008
实施例20 人源化抗体的体内中和活性评价
具体实验步骤参照实施例7，每个实验组10只BALB/C小鼠。
如图16所示，结果表明，与对照组(脾脏重量80.55mg)相比IL-33抗原组脾脏明显增重，为176.49mg。864F3-Hu4、864F3-Hu5、871G1-Hu1、871G1-Hu2、874F7-Hu1和874F7-Hu2抗体给药组的脾脏重量平均值分别为78.39mg、86.6mg、92.82mg、98.87mg、75.63mg和77.49mg，可见各人源化抗体在体内均具有明显的中和活性。
实施例21 人源化抗体的体内药效活性测定
实验方法参照实施例7，实验结果如图17所示，腹腔注射人IL-33后，可明显刺激小鼠脾脏肿大(IL-33组)，脾脏重量约为194.6mg，对照组(未给与人IL-33刺激)为76.1mg，而当分别给予5mg/kg的864F3-Hu4和874F7-Hu1进行单次治疗时可有效抑制小鼠脾脏肿大，脾脏重量分别为65.4mg和55.9mg。
上述实验结束时，对各组小鼠进行安乐死，并分别进行外周血中的嗜酸性粒细胞和IL-5水平检测。
将上述小鼠全血取出一部分置于普通EP管中，4℃放置5h以上使其完全凝血。凝血后将EP管置于4℃预冷的离心机中以8000rmp离心6min。离心后取出血清，置于新的离心管中，再次在4℃预冷的离心机中以8000rmp离心6min。离心后取出上清，分装，置于-80中保存。按照BD CBA Mouse Enhanced Sensivity Master Buffer Kit(购自BD biosciences，货号562246)的说明书要求对标准品和待测血清样品进行稀释。随后按照Mouse IL-5 Enhanced Sensitivity Flex Set(购自BD biosciences，货号562234)说明书要求对各样品进行处理，设置好检测参数在流式仪(BD FACSCelesta)对各样品进行检测，通过GraphPad Prism对上述采集到的数据进行分析，根据标准曲线计算出各实验组血清样品中IL-5的含量。
实验结果如图18所示，与对照组相比(未给与人IL-33刺激)，腹腔注射人IL-33后，可明显刺激小鼠外周血中IL-5的分泌(IL-33组)，约78.6倍，而当分别给予5mg/kg的864F3-Hu4和874F7-Hu1进行单次治疗时可有效抑制小鼠外周血IL-5分泌，分别为对照组的1.7倍和0.9倍。
将上述小鼠全血取出一部分置于EDTA抗凝管中，上下颠倒均匀，置于4℃ 备用。取10mL圆底离心管，向其中每管加入2μL mouse Fc blocker(购自BD biosciences，货号553142)。每个样品取50μL血样于10mL离心管中，轻轻吹打使血样与blocker混合均匀。置于冰上孵育15min。向每个离心管中加入2μL混合好的三种抗体：0.5μL CD45.2 Monoclonal Antibody(104),PerCP-Cyanine5.5(购自eBioscience TM，货号45-0454-82)、0.5μL CD170(Siglec F)Monoclonal Antibody(1RNM44N),PE(购自eBioscience TM，货号12-1702-82)、1μL Ly-6G/Ly-6C Monoclonal Antibody(RB6-8C5),APC-eFluor 780(购自eBioscience TM，货号47-5931-82)；冰上避光孵育1h。向离心管中加入500mL 1x Lysing buffer(购自BD Biosciences，货号555899)，混匀，室温裂解红细胞5min。将离心管进行350g离心5min。弃掉上清，向管中加入3mL预冷的PBS洗涤细胞，置于350g离心5min。弃掉上清，向管中加入500μL预冷、过滤处理的PBS，轻轻弹起沉降的细胞。将细胞悬液用细胞筛网过滤至流式管中，通过流式仪对各样品中嗜酸性粒细胞占白细胞的比例进行分析。
实验结果如图19所示，与对照组相比(未给与人IL-33刺激)，腹腔注射人IL-33后，可明显刺激小鼠外周血中嗜酸性粒细胞的增加(IL-33组)，约10.5倍，而当分别给予5mg/kg的864F3-Hu4和874F7-Hu1进行单次治疗时可有效降低小鼠外周血中由人IL-33刺激引起的嗜酸性粒细胞的增加，分别为对照组的1.6倍和1.58倍。
实施例22 人源化抗体对IL-33的结合表位测定
根据IL-33成熟蛋白的氨基酸序列：
MSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET(SEQ ID NO.55)(氨基酸序列来源于NCBI:NP_254274.1的第112位Ser至270位Thr)，合成如下16条多肽以进行人源化抗体对IL-33的结合表位表征，各多肽氨基酸序列如表4所示，其N-端均进行了biotin化标记。表4：多肽氨基酸序列
Figure PCTCN2021120785-appb-000009
Figure PCTCN2021120785-appb-000010
为了测定抗体对各多肽片段的结合活性，预先以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释生物素-亲和素(SA)至2μg/mL，100μL/孔，包被ELISA板，4℃，过夜；PBST洗涤3次，再用PBS配制的2％的BSA，于室温封闭2h，200μL/孔。PBST洗涤1次，分别加入终浓度为10μg/mL的864F3和874F7，100μL/孔，室温孵育1h。PBST洗涤3次，加入HRP标记的羊抗小鼠IgG二抗，室温孵育30min；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μL的TMB显色液，显色至合适深浅；每孔加入50μL 2M H 2SO 4终止液终止底物反应，于酶标仪450nm处读取OD值，分析待测抗体与各多肽片段的结合能力。实验结果如表5所示，可见874F7能显著的与Pep6结合而不与其他肽段结合。
表5：多肽氨基酸序列
Figure PCTCN2021120785-appb-000011
Figure PCTCN2021120785-appb-000012
基于上述实验结果，以及874F7-Hu1与鼠IL-33蛋白的结合Kd值为3.32×10 -8；，根据上述IL-33成熟蛋白的氨基酸序列选取：
“KKDEKKDKVLLSYYESQHPSNESGDGVDGK”区域(SEQ ID NO.55的41位-70位)通过PCR(聚合酶链式反应)进行丙氨酸扫描式定点突变，随后进行原核表达，并纯化，分别获得如下氨基酸位点突变的IL-33突变体蛋白：
K41A、K42A、D43A、E44A、K45A、K46A、D47A、K48A、V49A、L50A、L51A、S52A、Y53A、Y54A、E55A、S56A、Q57A、H58A、P59A、S60A、N61A、E62A、S63A、D65A、V67A、D68A、K70A。
随后参照实施例9的实验方法，分别测定上述各突变体蛋白对874F7-Hu1的亲和力，同时设置未进行任何突变的IL-33蛋白(WT-1、WT-2、WT-3、WT-4)作为对照。
代表性实验结果分别如图20-图23及表6所示，可见与WT相比，K45、V49、L50、D65突变后，874F7-Hu1与IL-33的亲和力显著减弱，下降25倍以上；S60、S52突变后，874F7-Hu1与IL-33的亲和力明显减弱，下降10-25倍；K48、L51、Y53、E55突变后，874F7-Hu1与IL-33的亲和力有一定减弱，下降2.5-10倍。这也说明影响874F7-Hu1与IL-33结合的关键位点主要为K45、V49、D65、L50，其次为S60、S52，再者为K48、L51、Y53、E55。
上述各位点在IL-33结晶3D结构图(来源于PDB:2KLL)中的位置如图24所示，这也进一步说明874F7-Hu1与IL-33的结合表位为包括上述关键氨基酸位点在内的线性和空间表位。
表6：874F7-Hu1与IL-33各突变体蛋白结合亲和力
Figure PCTCN2021120785-appb-000013
Figure PCTCN2021120785-appb-000014
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (25)

  1. 结合人IL-33的抗体或其抗原结合片段，其特征在于，其中该抗体或其抗原结合片段与人IL-33结合的亲和力EC 50小于1nM。
  2. 如权利要求1所述的结合人IL-33的抗体或其抗原结合片段，其特征在于，所述抗体的轻链具有SEQ ID No:22所示的L-CDR2，并且具有以下特征：
    (t1)阻断IL-33和受体ST2的结合；
    (t2)抑制IL-33诱导的HUVEC细胞的IL-6分泌；
    (t3)抑制IL-33蛋白诱导人PBMC分泌IFNγ；
    (t4)抑制IL-33诱导NK细胞分泌IFNγ；和
    (t5)抑制IL-33诱导KU812细胞分泌IL-5和IL13。
  3. 如权利要求1或2所述的结合人IL-33的抗体或其抗原结合片段，其特征在于，包括：
    (a)重链互补决定区H-CDR1、H-CDR2、H-CDR3，所述H-CDR1、H-CDR2、H-CDR3的氨基酸序列分别如SEQ ID NO：15、16和17所示，或者分别如SEQ ID NO：18、19和20所示，或者分别如SEQ ID NO：18、24和25所示，和
    (b)轻链互补决定区L-CDR1、L-CDR2、L-CDR3，所述L-CDR1、L-CDR2、L-CDR3的氨基酸序列分别如SEQ ID NO：21、22和23所示，或者分别如SEQ ID NO：26、22和27所示，或者分别如SEQ ID NO：26、22和28所示。
  4. 如权利要求1或2所述的结合人IL-33的抗体或其抗原结合片段，其特征在于，所述的抗体为鼠源抗体、嵌合抗体或人源化抗体。
  5. 如权利要求1或2所述的结合人IL-33的抗体或其抗原结合片段，其特征在于，所述的抗原结合片段包括Fab片段、F(ab’) 2片段、Fv片段。
  6. 如权利要求3所述的结合人IL-33的抗体或其抗原结合片段，其特征在于，所述的结合人IL-33的抗体或其抗原结合片段的重链可变区和轻链可变区的氨基酸序列分别如SEQ ID NO：2和SEQ ID NO：6所示，或者分别如SEQ ID NO：4和SEQ ID NO：6所示，或者分别如SEQ ID NO：8和SEQ ID NO：10所示，或者分别如SEQ ID NO：4和SEQ ID NO：12所示。
  7. 如权利要求3所述的结合人IL-33的抗体或其抗原结合片段，其特征在于，所述的结合人IL-33的抗体或其抗原结合片段的轻链可变区的氨基酸序列如 SEQ ID NO：32所示，重链可变区的氨基酸序列如SEQ ID NO：29、30、31、33、34或35所示。
  8. 如权利要求3所述的结合人IL-33的抗体或其抗原结合片段，其特征在于，所述的结合人IL-33的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：40所示，重链可变区的氨基酸序列如SEQ ID NO：37、38或39所示。
  9. 如权利要求3所述的结合人IL-33的抗体或其抗原结合片段，其特征在于，所述的结合人IL-33的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：36所示，重链可变区的氨基酸序列如SEQ ID NO：33、34或35所示。
  10. 如权利要求1或2所述的结合人IL-33的抗体或其抗原结合片段，其特征在于，所述抗体的重链恒定区选自人IgG1、IgG2、IgG3或IgG4的重链恒定区。
  11. 如权利要求10所述的结合人IL-33的抗体或其抗原结合片段，其特征在于，所述的结合人IL-33的抗体或其抗原结合片段的重链恒定区和轻链恒定区的氨基酸序列分别如SEQ ID NO：13和SEQ ID NO：14所示。
  12. 如权利要求10所述的结合人IL-33的抗体或其抗原结合片段，其特征在于，所述的结合人IL-33的抗体或其抗原结合片段与IL-33蛋白的结合表位包含对应于SEQ ID NO.55的选自下组的位点：
    第45位赖氨酸(K45)、第49位缬氨酸(V49)、第65位天冬氨酸(D65)、第50位亮氨酸(L50)、第60位丝氨酸(S60)、第52位丝氨酸(S52)、第48位赖氨酸(K48)、第51位亮氨酸(L51)、第53位酪氨酸(Y53)、第55位谷氨酸(E55)。
  13. 一种核苷酸分子，其特征在于，所述核苷酸分子编码如权利要求1-12中任一项所述的结合人IL-33的抗体或其抗原结合片段。
  14. 如权利要求13所述的核苷酸分子，其特征在于，所述核苷酸分子编码重链可变区和轻链可变区的核苷酸序列分别如SEQ ID NO：1和SEQ ID NO：5所示所示，或者分别如SEQ ID NO：3和SEQ ID NO：5所示，或者分别如SEQ ID NO：7和SEQ ID NO：9所示，或者分别如SEQ ID NO：3和SEQ ID NO： 11所示。
  15. 如权利要求13所述的核苷酸分子，其特征在于，所述核苷酸分子编码重链可变区的核苷酸序列分别如SEQ ID NO：41、42、43、45、46或47所示，编码轻链可变区的核苷酸序列如SEQ ID NO：44所示。
  16. 如权利要求13所述的核苷酸分子，其特征在于，所述核苷酸分子编码重链可变区的核苷酸序列分别如SEQ ID NO：49、50或51所示，编码轻链可变区的核苷酸序列如SEQ ID NO：52所示。
  17. 如权利要求13所述的核苷酸分子，其特征在于，所述核苷酸分子编码重链可变区的核苷酸序列分别如SEQ ID NO：45、46或47所示，编码轻链可变区的核苷酸序列如SEQ ID NO：48所示。
  18. 一种表达载体，其特征在于，所述表达载体含有如权利要求13-17中任一项所述的核苷酸分子。
  19. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求18所述的表达载体。
  20. 一种制备如权利要求1-12中任一项所述的结合人IL-33的抗体或其抗原结合片段的方法，其特征在于，所述方法包括以下步骤：
    a)在表达条件下，培养如权利要求19所述的宿主细胞，从而表达所述的结合人IL-33的抗体或其抗原结合片段；
    b)分离并纯化a)所述的结合人IL-33的抗体或其抗原结合片段。
  21. 一种组合物，其特征在于，所述组合物含有如权利要求1-12中任一项所述的结合人IL-33的抗体或其抗原结合片段和药学上可接受的载体。
  22. 一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物含有：
    (a)抗体部分，所述抗体部分包含如权利要求1-12中任一项所述的结合人IL-33的抗体或其抗原结合片段；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
  23. 如权利要求1-12中任一项所述的结合人IL-33的抗体或其抗原结合片段或如权利要求21所述的组合物、如权利要求22所述的抗体药物偶联物在制备治疗哮喘、关节炎、特异反应性/过敏性皮炎、慢性鼻窦炎、慢性阻塞性肺病 (COPD)、系统性硬化症、肝纤维化、牛皮癣、溃疡性结肠炎、克罗恩氏病、多发性硬化症、糖尿病肾脏疾病、发炎性肠道疾病、银屑病、嗜酸性食管炎、糖尿病性黄斑水肿、年龄相关性黄斑变性、干眼病、肿瘤的药物中的应用。
  24. 一种治疗哮喘、关节炎、特异反应性/过敏性皮炎、慢性鼻窦炎、慢性阻塞性肺病(COPD)、系统性硬化症、肝纤维化、牛皮癣、溃疡性结肠炎、克罗恩氏病、多发性硬化症、糖尿病肾脏疾病、发炎性肠道疾病、银屑病、嗜酸性食管炎、糖尿病性黄斑水肿、年龄相关性黄斑变性、干眼病、肿瘤的方法，其特征在于，给需要的对象施用如权利要求1-12中任一项所述的结合人IL-33的抗体或其抗原结合片段、如权利要求21所述的组合物或如权利要求22所述的药物偶联物、或其组合。
  25. 一种IL-33蛋白的突变体，其特征在于，对应于野生型人IL-33蛋白的氨基酸序列，所述突变体包含对应于SEQ ID NO.55的选自下组的一个或多个位点发生突变：
    (Z1)第45位赖氨酸(K45)；
    (Z2)第49位缬氨酸(V49)；
    (Z3)第65位天冬氨酸(D65)；
    (Z4)第50位亮氨酸(L50)；
    (Z5)第60位丝氨酸(S60)；
    (Z6)第52位丝氨酸(S52)；
    (Z7)第48位赖氨酸(K48)；
    (Z8)第51位亮氨酸(L51)；
    (Z9)第53位酪氨酸(Y53)；
    (Z10)第55位谷氨酸(E55)。
PCT/CN2021/120785 2020-09-25 2021-09-26 结合人il-33的抗体、其制备方法和用途 WO2022063281A1 (zh)

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