Antibodies that bind to human IL-33, methods for preparing the same, and uses thereof

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof that binds to human IL-33, can bind to human IL-33 in a high-affinity manner, blocks the binding of ST2 to IL-33, and is applied to the preparation of drugs for treating diseases with high expression of IL-33 (such as asthma, atopic / allergic dermatitis, chronic rhinosinusitis, chronic obstructive pulmonary disease (COPD), etc.), and has good clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to an antibody that binds to human IL-33, a method for preparing the same, and uses thereof. Background Art

[0002] Interleukin-33 (IL-33) is a multifunctional cytokine and 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 to express by inflammatory factors. Studies have found that IL-33 is a bifunctional protein. On the one hand, IL-33 is localized in the nucleus and functions as a transcription factor; on the other hand, IL-33 is secreted extracellularly and functions as a cytokine by interacting with its receptor ST2. As a cytokine, IL-33 is a TH-2 type cytokine and is considered an alarmin. By binding to ST2, it induces TH-2 cells to secrete TH-2 type cytokines such as IL-4, IL-5, and IL-13. In addition, IL-33 can also cause mast cells and basophils to secrete inflammatory cytokines and chemokines, such as IL-1β, IL-6, IL-8, TNFα, etc., and lead to NK cells and NKT cells secreting TH-1 type cytokines, such as IFNγ, etc.

[0003] Asthma is also known as bronchial asthma. Bronchial asthma is a chronic airway inflammation involving multiple cell types and cell components. Asthma has always been considered an airway inflammation driven by CD4+ Th-2 cells. However, anti-CD4 antibodies almost completely deplete CD4+ cells but do not completely reduce the production of IL-4, IL-5, or IL-13 in the lungs of asthmatic mice, indicating that there must be other cellular sources of these Th-2 cytokines. IL-33 can produce IL-5 and IL-13 from ILC2s expressing ST2, suggesting that IL-5-induced eosinophilia and IL-13-induced mucus production can be induced even in the absence of Th-2 cells. In addition, the expression level of IL-33 in the lungs of asthma patients is higher than that of healthy individuals, and its expression is particularly obvious in the lung tissues of severe asthma patients. IL-33 promotes collagen synthesis in asthmatic fibroblasts in children with severe asthma, suggesting that IL-33 plays a certain role in the occurrence and development of characteristic airway remodeling in severe asthma. Allergic airway inflammation can be alleviated by treatment with anti-IL-33 antibodies. Since 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. These findings indicate that IL-33 can coordinate the bridge between innate immunity and adaptive immunity, thus developing into a severe asthma phenotype. In addition to asthma, the IL-33 pathway is also involved in the treatment of various diseases, such as atopic / allergic dermatitis, arthritis, chronic rhinosinusitis, 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, etc. The present invention provides an IL-33 inhibitor that binds to IL-33 in a high-affinity manner and effectively neutralizes the activity of IL-33. Summary of the Invention

[0004] To solve the above technical problems, the inventors of the present invention conducted a large number of experiments, from antigen immunization, hybridoma screening, antibody expression and purification to bioactivity identification, and screened and obtained a murine antibody that specifically binds to human IL-33. On this basis, its chimeric antibody and humanized antibody were further constructed.

[0005] Accordingly, the object of the present invention is to provide an antibody or antigen-binding fragment thereof that binds to human IL-33; to provide a nucleotide molecule encoding the antibody or antigen-binding fragment thereof that binds to human IL-33; to provide an expression vector containing the nucleotide molecule; to provide a host cell of the expression vector; to provide a preparation method of the antibody or antigen-binding fragment thereof that binds to human IL-33; to provide a pharmaceutical composition containing the antibody or antigen-binding fragment thereof that binds to human IL-33; to provide the use of the antibody or antigen-binding fragment thereof that binds to human IL-33 in the preparation of a drug.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] On the one hand, the present invention provides an antibody or antigen-binding fragment thereof that binds to human IL-33, wherein the affinity EC of the antibody or antigen-binding fragment thereof for binding to human IL-33 50 is less than 1 nM.

[0008] In another preferred embodiment, the light chain of the antibody has L-CDR2 shown in SEQ ID No: 22 and has the following characteristics:

[0009] (t1) Block the binding of IL-33 and the receptor ST2;

[0010] (t2) Inhibit the secretion of IL-6 by HUVEC cells induced by IL-33;

[0011] (t3) Inhibit the secretion of IFNγ by human PBMC induced by IL-33 protein;

[0012] (t4) Inhibit the secretion of IFNγ by NK cells induced by IL-33; and

[0013] (t5) Inhibit the secretion of IL-5 and IL13 by KU812 cells induced by IL-33.

[0014] In another preferred embodiment, the Kd value of the antibody for human IL33 is much less than the Kd value for mouse IL33 (the difference is 20 times or more).

[0015] In another preferred embodiment, the antibody includes:

[0016] (a) Heavy chain complementary determining regions H-CDR1, H-CDR2, H-CDR3, wherein H-CDR1 is as shown in SEQ ID NO: 18 or a mutant of SEQ ID NO: 18 with at most 2 amino acid substitution mutations; H-CDR2 is as shown in SEQ ID NO: 19 or a mutant of SEQ ID NO: 19 with at most 4 amino acid substitution mutations; the amino acid sequence of H-CDR3 is as shown in SEQ ID NO: 20 or a mutant of SEQ ID NO: 20 with at most 7 amino acid substitution mutations, and

[0017] (b) Light chain complementary determining regions L-CDR1, L-CDR2, L-CDR3, wherein the amino acid sequence of L-CDR1 is as shown in SEQ ID NO: 21 or a mutant of SEQ ID NO: 21 with at most 2 amino acid substitution mutations, the amino acid sequence of L-CDR2 is as shown in SEQ ID NO: 22, and the amino acid sequence of L-CDR3 is as shown in SEQ ID NO: 23 or a mutant of SEQ ID NO: 23 with at most 3 amino acid substitution mutations. As a preferred embodiment, the antibody or antigen-binding fragment thereof that binds to human IL-33 comprises:

[0018] (a) Heavy chain complementary determining regions H-CDR1, H-CDR2, H-CDR3, wherein the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are respectively as shown in SEQ ID NO: 15, 16, and 17, or respectively as shown in SEQ ID NO: 18, 19, and 20, or respectively as shown in SEQ ID NO: 18, 24, and 25, and

[0019] (b) Light chain complementary determining regions L-CDR1, L-CDR2, L-CDR3, wherein the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are respectively as shown in SEQ ID NO: 21, 22, and 23, or respectively as shown in SEQ ID NO: 26, 22, and 27, or respectively as shown in SEQ ID NO: 26, 22, and 28.

[0020] In another preferred example, the antibody or antigen-binding fragment thereof that binds to human IL-33 comprises:

[0021] (a1) Heavy chain complementary determining regions H-CDR1, H-CDR2, H-CDR3, wherein the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are respectively as shown in SEQ ID NO: 15, 16, and 17, and

[0022] (b1) Light chain complementary determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are shown in SEQ ID NO: 21, 22, and 23 respectively; or

[0023] (a2) Heavy chain complementary determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are shown in 18, 19, and 20 respectively, and

[0024] (b2) Light chain complementary determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are shown in SEQ ID NO: 21, 22, and 23 respectively; or

[0025] (a3) Heavy chain complementary determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are shown in SEQ ID NO: 18, 24, and 25 respectively, and

[0026] (b3) Light chain complementary determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are shown in SEQ ID NO: 26, 22, and 27 respectively; or

[0027] (a4) Heavy chain complementary determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are shown in SEQ ID NO: 18, 19, and 20 respectively, and

[0028] (b4) Light chain complementary determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are shown in SEQ ID NO: 26, 22, and 28 respectively.

[0029] In another preferred example, the antibody or antigen-binding fragment thereof that binds to human IL-33 includes:

[0030] (a2) Heavy chain complementary determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are shown in 18, 19, and 20 respectively, and

[0031] (b2) Light chain complementary determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are shown in SEQ ID NO: 21, 22, and 23 respectively; or

[0032] (a3) Heavy chain complementary determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are shown in SEQ ID NO: 18, 24, and 25 respectively, and

[0033] (b3) Light chain complementary determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are shown in SEQ ID NO: 26, 22, and 27 respectively.

[0034] In another preferred embodiment, the amino acid sequence of any of the above CDRs contains a derived CDR sequence with 1, 2, 3, 4, 5, 6, or 7 amino acids added, deleted, modified, and / or substituted, and the derived antibody formed by VH and VL containing the derived CDR sequence can retain the affinity for binding to IL-33.

[0035] The "antibody (Ab)" of the present invention is a heterotetrameric glycoprotein of approximately 150,000 daltons, which is 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 between heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. The antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed by at least two antibodies (such as bispecific antibodies), etc.

[0036] The "monoclonal antibody" of the present invention refers to an antibody obtained from a substantially homogeneous population, that is, the individual antibodies contained in the population are the same, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, different from conventional polyclonal antibody preparations (usually having different antibodies against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of the antibody, which is obtained from a substantially homogeneous group of antibodies, and this should not be interpreted as requiring any special method to produce the antibody.

[0037] The "antigen-binding fragment" of the present invention refers to a fragment of an antibody that can specifically bind to human IL-33. Examples of the antigen-binding fragment of the present invention include Fab fragments, F(ab’)2 fragments, Fv fragments, etc. The Fab fragment is a fragment produced by digesting an antibody with papain. The F(ab’)2 fragment is a fragment produced by digesting an antibody with pepsin. The Fv fragment is composed of a dimer in which the variable region of the heavy chain and the variable region of the light chain of the antibody are closely non-covalently associated.

[0038] As a preferred embodiment, the antibody is a murine antibody, a chimeric antibody or a humanized antibody.

[0039] 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 immunizing a mouse with human IL-33 as an antigen and screening hybridoma cells.

[0040] The "chimeric antibody" of the present invention refers to an antibody that contains the variable region sequences of the heavy chain and the light chain derived from one species and the constant region sequences derived from another species. For example, an antibody having murine heavy and light chain variable regions linked to a human constant region. Preferably, the chimeric antibody of the present invention is obtained by recombining the heavy chain variable regions of murine antibodies 864F3, 874F7, 871G1 with the human IgG1, IgG2, IgG3 or IgG4 heavy chain constant regions containing mutations, and recombining the light chain variable region with the human kappa chain constant region.

[0041] The "humanized antibody" of the present invention refers to an antibody whose CDRs are derived from antibodies of non-human species (preferably mice), and the remaining parts of the antibody molecule (including the framework regions and the constant regions) are derived from human antibodies. In addition, framework region residues can be altered to maintain binding affinity. Preferably, the humanized antibody of the present invention is obtained by recombining the CDR regions of murine antibodies 864F3, 874F7, 871G1 and the non-CDR regions derived from human antibodies, recombining the heavy chain variable region with the human IgG1, IgG2, IgG3 or IgG4 heavy chain constant regions containing mutations, recombining the light chain variable region with the human kappa chain constant region, and mutating some residues that have an important impact.

[0042] As a preferred embodiment, the antigen-binding fragment includes Fab fragments, F(ab’)2 fragments, Fv fragments.

[0043] As a preferred embodiment, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment that binds to human IL-33 are shown as SEQ ID NO: 2 and SEQ ID NO: 6, respectively, or as 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.

[0044] As a preferred embodiment, 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 as SEQ ID NO: 32, and the amino acid sequences of the heavy chain variable region are shown as SEQ ID NO: 29, 30, 31, 33, 34 or 35.

[0045] As a preferred embodiment, 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 as SEQ ID NO: 40, and the amino acid sequences of the heavy chain variable region are shown as SEQ ID NO: 37, 38 or 39.

[0046] As a preferred embodiment, the heavy chain constant region of the antibody is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4.

[0047] As a preferred embodiment, 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 as SEQ ID NO: 36, and the amino acid sequences of the heavy chain variable region are shown as SEQ ID NO: 33, 34 or 35.

[0048] In another preferred example, 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 as SEQ ID NO: 32, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 34; or 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 as SEQ ID NO: 40, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 38. In another preferred example, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown as SEQ ID NO: 32, 40 or 36 in the sequence listing.

[0049] In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO: 29, 30, 31, 33, 34, 35, 37, 38 or 39 in the sequence listing.

[0050] As a preferred embodiment, the amino acid sequences of the heavy chain constant region and the light chain constant region of the antibody or its antigen-binding fragment that binds to human IL-33 are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

[0051] In another preferred embodiment, the binding epitope of the antibody or its antigen-binding fragment that binds to human IL-33 and the IL-33 protein contains sites selected from the following groups corresponding to SEQ ID NO.55:

[0052] Lysine at position 45 (K45), valine at position 49 (V49), aspartic acid at position 65 (D65), leucine at position 50 (L50), serine at position 60 (S60), serine at position 52 (S52), lysine at position 48 (K48), leucine at position 51 (L51), tyrosine at position 53 (Y53), glutamic acid at position 55 (E55);

[0053] More preferably, it contains sites selected from the following groups: lysine at position 45 (K45), valine at position 49 (V49), aspartic acid at position 65 (D65), leucine at position 50 (L50).

[0054] In another preferred embodiment, the site corresponds to the amino acid sequence of wild-type human IL-33 protein, and the amino acid sequence is Ser at position 112 to Thr at position 270 of NCBI: NP_254274.1.

[0055] On the other hand, the present invention provides a nucleotide molecule that encodes the above-mentioned antibody or its antigen-binding fragment that binds to human IL-33.

[0056] As a preferred embodiment, the nucleotide sequences encoding the heavy chain variable region and the light chain variable region are shown in SEQ ID NO: 1 and SEQ ID NO: 5, respectively, or are shown in SEQ ID NO: 3 and SEQ ID NO: 5, respectively, or are shown in SEQ ID NO: 7 and SEQ ID NO: 9, respectively, or are shown in SEQ ID NO: 3 and SEQ ID NO: 11, respectively.

[0057] As a preferred embodiment, the nucleotide sequences encoding the heavy chain variable regions of the nucleotide molecules are respectively as shown in SEQ ID NO: 41, 42, 43, 45, 46 or 47, and the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO: 44.

[0058] As a preferred embodiment, the nucleotide sequences encoding the heavy chain variable regions of the nucleotide molecules are respectively as shown in SEQ ID NO: 49, 50 or 51, and the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO: 52.

[0059] As a preferred embodiment, the nucleotide sequences encoding the heavy chain variable regions of the nucleotide molecules are respectively as shown in SEQ ID NO: 45, 46 or 47, and the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO: 48.

[0060] The preparation method of the nucleotide molecule of the present invention is a conventional preparation method in the art. Preferably, it includes the following preparation methods: obtaining the nucleotide molecule encoding the above monoclonal antibody through gene cloning techniques such as PCR method, or obtaining the nucleotide molecule encoding the above monoclonal antibody through the method of artificial total sequence synthesis.

[0061] Those skilled in the art know that the nucleotide sequence encoding the amino acid sequence of the antibody or its antigen-binding fragment that binds to human IL-33 can be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide a homolog of the polynucleotide. The homolog of the polynucleotide in the present invention can be prepared by substituting, deleting or adding one or more bases of the gene encoding the antibody or its antigen-binding fragment that binds to human IL-33 within the range of maintaining the antibody activity.

[0062] On the other hand, the present invention provides an expression vector, and the expression vector contains the above nucleotide molecule.

[0063] The expression vector described above is a conventional expression vector in the art, which refers to an expression vector containing appropriate regulatory sequences, such as a promoter sequence, a terminator sequence, a polyadenylation sequence, an enhancer sequence, a marker gene and / or sequence, and other appropriate sequences. The expression vector can be a virus or a plasmid, such as an appropriate phage or phagemid. For more technical details, please refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and protocols for nucleic acid manipulation can be found in Current Protocols in Molecular Biology, Second Edition, edited by Ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, pcDNA4, pDHFF, pGM-CSF or pCHO1.0.

[0064] The present invention further provides a host cell containing the above-mentioned expression vector.

[0065] The host cell of the present invention is various conventional host cells in the art, as long as it can satisfy the stable self-replication of the above-mentioned recombinant expression vector and the effective expression of the carried nucleotide. The host cells include prokaryotic expression cells and eukaryotic expression cells. The host cells preferably include: COS, CHO (Chinese Hamster Ovary), NS0, sf9, sf21, DH5α, BL21(DE3) or TGi, more preferably E. coli TGi, BL21(DE3) cells (expressing single-chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies). Transforming the aforementioned expression vector into the host cell can obtain the preferred recombinant expression transformant of the present invention. The transformation method is a conventional transformation method in the art, preferably chemical transformation, heat shock method or electroporation method.

[0066] On the other hand, the present invention provides a method for the antibody or its antigen-binding fragment that binds to human IL-33 as described above, characterized in that the method comprises the following steps:

[0067] a) Culturing the above-mentioned host cell under expression conditions to express the antibody or its antigen-binding fragment that binds to human IL-33;

[0068] b) Separating and purifying the antibody or its antigen-binding fragment that binds to human IL-33 obtained in a).

[0069] The culturing method of the host cell and the separation and purification method of the antibody described in 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 or its antigen-binding fragment that binds to human IL-33 disclosed in the present invention includes: culturing the above-mentioned host cell under expression conditions to express the antibody or its antigen-binding fragment that binds to human IL-33; separating and purifying the antibody or its antigen-binding fragment that binds to human IL-33. By using the above method, the recombinant protein can be purified into a substantially homogeneous substance, such as a single band on SDS-PAGE electrophoresis.

[0070] The antibody or its antigen-binding fragment that binds to human IL-33 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, changing pH, etc. can be used to elute the antibody or its antigen-binding fragment that binds to human IL-33 bound to the affinity column. The inventors of the present invention conducted detection experiments on the obtained antibody or its antigen-binding fragment that binds to human IL-33. The experimental results show that the antibody or its antigen-binding fragment that binds to human IL-33 can bind well to the antigen and has a high affinity.

[0071] On the other hand, the present invention provides a composition, which contains the above-mentioned antibody or its antigen-binding fragment that binds to human IL-33 and a pharmaceutically acceptable carrier.

[0072] The antibody or its antigen-binding fragment 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 to exert its efficacy more stably. These preparations can ensure the conformational integrity of the antibody or its antigen-binding fragment that binds to human IL-33 disclosed in the present invention, and at the same time protect the multiple functional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation). Usually, for liquid preparations, they can be stored stably at least for one year under the condition of 2°C - 8°C, and for lyophilized preparations, they remain stable at 30°C for at least six months. The bispecific antibody preparation can be common preparations such as suspension, aqueous injection, lyophilization, etc. in the pharmaceutical field.

[0073] For the aqueous or lyophilized preparation of the antibody or its antigen-binding fragment that binds to human IL-33 disclosed in the present invention, pharmaceutically acceptable carriers preferably include, but are not limited to: one or a combination of surfactants, solution stabilizers, isotonicity regulators, and buffers. Among them, surfactants preferably include, but are not limited to: non-ionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; myristyl, linoleyl, or octadecyl sarcosine; Pluronics; MONAQUATTM, etc., and the addition amount thereof should minimize the granulation tendency of the antibody or its antigen-binding fragment that binds to human IL-33. Solution stabilizers preferably include, but are not limited to, one or a combination of the following: saccharides, for example, reducing sugars and non-reducing sugars; amino acids, for example, monosodium glutamate or histidine; alcohols, for example: trihydric alcohols, higher polyhydric alcohols, propylene glycol, polyethylene glycol, etc., and the addition amount of the solution stabilizer should keep the finally formed preparation in a stable state within the time considered stable by those skilled in the art. Isotonicity regulators preferably include, but are not limited to, one or a combination of sodium chloride and mannitol. Buffers preferably include, but are not limited to: one or a combination of Tris, histidine buffer, and phosphate buffer.

[0074] On the other hand, the present invention provides an antibody-drug conjugate, and the antibody-drug conjugate contains:

[0075] (a) an antibody moiety, the antibody moiety comprising the above-mentioned antibody or its antigen-binding fragment that binds to human IL-33; and

[0076] (b) a conjugate moiety conjugated to the antibody moiety, the conjugate moiety being selected from the group consisting of: detectable labels, drugs, toxins, cytokines, radionuclides, enzymes, or a combination thereof.

[0077] On the other hand, the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment that binds to human IL-33, or the pharmaceutical composition or antibody-drug conjugate in the preparation of drugs for treating asthma, arthritis, atopic / allergic dermatitis, chronic rhinosinusitis, 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, and tumors. The arthritis includes rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gouty arthritis, reactive arthritis, infectious arthritis, traumatic arthritis, psoriatic arthritis, and enteropathic arthritis.

[0078] When the antibody or its antigen-binding fragment that binds to human IL-33 and its composition of the present invention are administered to animals including humans, the dosage varies depending on the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. The total dosage should not exceed a certain range with reference to the results of animal experiments and various circumstances. Specifically, the dosage for intravenous injection is 1 - 1800 mg / day.

[0079] On the other hand, the present invention provides a method for treating asthma, arthritis, atopic / allergic dermatitis, chronic rhinosinusitis, 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, which is characterized by administering to a subject in need the above-mentioned antibody or its antigen-binding fragment that binds to human IL-33, or a pharmaceutical composition, an antibody-drug conjugate, or a combination thereof.

[0080] On the other hand, the present invention provides a mutant of the IL-33 protein, corresponding to the amino acid sequence of the wild-type human IL-33 protein, wherein the mutant contains one or more sites selected from the following groups mutated;

[0081] (Z1) Lysine at position 45 (K45);

[0082] (Z2) Valine at position 49 (V49);

[0083] (Z3) Aspartic acid at position 65 (D65);

[0084] (Z4) Leucine at position 50 (L50);

[0085] (Z5) Serine at position 60 (S60);

[0086] (Z6) Serine at position 52 (S52);

[0087] (Z7) Lysine at position 48 (K48);

[0088] (Z8) Leucine at position 51 (L51);

[0089] (Z9) Tyrosine at position 53 (Y53);

[0090] (Z10) Glutamic acid at position 55 (E55).

[0091] In another preferred embodiment, the sequence of the wild-type human IL-33 protein is as shown in SEQ ID NO.55.

[0092] In another preferred example, the mutant contains mutations at one or more sites selected from the following groups;

[0093] (Z1) Lysine at position 45 (K45);

[0094] (Z2) Valine at position 49 (V49);

[0095] (Z3) Aspartic acid at position 65 (D65);

[0096] (Z4) Leucine at position 50 (L50), and / or

[0097] The mutant contains mutations at one or more sites selected from the following groups;

[0098] (Z5) Serine at position 60 (S60);

[0099] (Z6) Serine at position 52 (S52); and / or

[0100] The mutant contains mutations at one or more sites selected from the following groups;

[0101] (Z7) Lysine at position 48 (K48);

[0102] (Z8) Leucine at position 51 (L51);

[0103] (Z9) Tyrosine at position 53 (Y53);

[0104] (Z10) Glutamic acid at position 55 (E55).

[0105] In another preferred example, compared with the affinity of wild-type IL-33 protein and an antibody that binds to human IL-33, the affinity of the mutant of the IL-33 protein for the antibody that binds to human IL-33 is decreased by 1-fold, preferably 5-fold, more preferably 10-fold, more preferably 25-fold, and most preferably 50-fold.

[0106] In another preferred example, the mutant further contains mutations at one or more sites selected from the following groups;

[0107] (Z11) Lysine at position 41 (K41);

[0108] (Z12) Lysine at position 42 (K42);

[0109] (Z13) Aspartic acid at position 43 (D43);

[0110] (Z14) Glutamic acid at position 44 (E44);

[0111] (Z15) Lysine at position 46 (K46);

[0112] (Z16) Aspartic acid at position 47 (D47);

[0113] (Z17) Tyrosine at position 54 (Y54);

[0114] (Z18) Serine at position 56 (S56);

[0115] (Z19) Glutamine at position 57 (Q57);

[0116] (Z20) Histidine at position 58 (H58);

[0117] (Z21) Proline at position 59 (P59);

[0118] (Z22) Asparagine at position 61 (N61);

[0119] (Z23) Glutamic acid at position 62 (E62);

[0120] (Z24) Serine at position 63 (S63);

[0121] (Z25) Valine at position 67 (V67);

[0122] (Z26) Aspartic acid at position 68 (D68);

[0123] (Z27) Lysine at position 70 (K70).

[0124] In another preferred example, the mutant of the IL-33 protein comprises mutating one or more amino acids in (Z1)-(Z27) to alanine (A) or glycine (G).

[0125] In another preferred example, the mutations in the mutant of the IL-33 protein are selected from the following group:

[0126] 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, or a combination thereof;

[0127] Preferably one or more of the following group: K45A, V49A, D65A, L50A, S60A, S52A, K48A, L51A, Y53A, E55A; More preferably one or more of the following group: K45A, V49A, D65A, L50A, S60A, S52A; Most preferably one or more of the following group: K45A, V49A, D65A, L50A.

[0128] In another preferred embodiment, the mutant of the IL-33 protein is selected from the group consisting of:

[0129] A derivative polypeptide having the function of binding to an anti-IL-33 antibody, which is formed by substituting, deleting or adding one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, and most preferably 1 amino acid residue in the amino acid sequence shown in SEQ ID NO.: 55.

[0130] In another preferred embodiment, the amino acid sequence of the mutant of the IL-33 protein has at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99% or more sequence identity compared with SEQ ID NO. 55.

[0131] On the other hand, the present invention provides a method for evaluating the binding epitope of an anti-human IL-33 antibody, comprising:

[0132] (S1) Providing an anti-human IL-33 antibody;

[0133] (S2) Detecting the affinity A1 of the anti-human IL-33 antibody for the IL-33 mutant compared with the affinity A2 for the wild-type IL-33 protein, wherein the IL-33 mutant comprises one or more site mutations of K45A, V49A, L50A, D65A, S60A or S52A. If the affinity decrease ratio A1 / A2 ≥ 2.5-fold, preferably ≥ 10-fold, it indicates that the linear and / or spatial epitope of the anti-human IL-33 antibody binding to the wild-type IL-33 protein comprises one or more of the sites K45, V49, L50, D65, S60A or S52A; wherein, the anti-human IL-33 antibody comprises:

[0134] Heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of H-CDR1, H-CDR2, H-CDR3 are respectively shown in SEQ ID NO: 18, 24 and 25, and

[0135] Light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequences of L-CDR1, L-CDR2, L-CDR3 are respectively shown in SEQ ID NO: 26, 22 and 27.

[0136] Based on the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0137] The reagents and raw materials used in the present invention are all commercially available.

[0138] The positive and progressive effects of the present invention are as follows: Currently, there is an urgent need in clinical practice to develop new, specific, and highly effective therapeutic drugs for diseases with strong IL-33 expression, so as to improve the quality of life of people suffering from such diseases and provide more and more effective treatment options 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 to treat various diseases, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1A ~B: Binding activity of murine antibody to human IL-33.

[0140] Figure 2A ~B: Activity of murine antibody in blocking the binding of ST2 to IL-33.

[0141] Figure 3A ~C: Inhibitory effect of murine antibody on IL-33-induced IL-6 expression in HUVEC cells.

[0142] Figure 4 : Inhibitory effect of murine antibody on IL-33-induced IL-8 expression in HUVEC cells.

[0143] Figure 5 : Cross-reactivity of murine antibody with cynomolgus monkey IL-33 antigen.

[0144] Figure 6 : Evaluation of in vivo neutralizing activity of murine antibody by spleen weight experiment.

[0145] Figure 7 : Binding activity of chimeric antibody to human IL-33.

[0146] Figure 8 : Activity of chimeric antibody in blocking the binding of ST2 to IL-33.

[0147] Figure 9A ~D: Binding activity of each humanized antibody to human IL-33.

[0148] Figure 10 : Activity of humanized antibody in blocking the binding of ST2 to IL-33.

[0149] Figure 11 : Inhibitory effect of humanized antibody on IL-33-induced IL-6 expression in HUVEC cells.

[0150] Figure 12 : Inhibitory effect of humanized antibody on IL-33-induced IFNγ secretion by PBMC.

[0151] Figure 13: Inhibitory effect of humanized antibody on IFNγ secretion by NK cells induced by IL-33.

[0152] Figure 14 : Humanized antibodies 864F3-Hu4 and 874F7-Hu1 can effectively inhibit IL-5 secretion by KU812 cells induced by IL-33.

[0153] Figure 15 : Humanized antibodies 864F3-Hu4 and 874F7-Hu1 can effectively inhibit IL-13 secretion by KU812 cells induced by IL-33.

[0154] Figure 16 : Evaluation of in vivo pharmacodynamic activity of humanized antibody by spleen weight experiment.

[0155] Figure 17 : Evaluation of in vivo pharmacodynamic activity of humanized antibodies 864F3-Hu4 and 874F7-Hu1 by spleen weight experiment.

[0156] Figure 18 : Humanized antibodies 864F3-Hu4 and 874F7-Hu1 can effectively inhibit IL-5 secretion in mouse peripheral blood.

[0157] Figure 19 : Humanized antibodies 864F3-Hu4 and 874F7-Hu1 can effectively reduce the increase in eosinophils in mouse peripheral blood caused by human IL-33 stimulation.

[0158] Figure 20 : Affinity of IL-33 mutant proteins (E44A, K45A, K46A, Q57A, H58A, P59A, S60A) for 874F7-Hu1.

[0159] Figure 21 : Affinity of IL-33 mutant proteins (K48A, V49A, L51A, S52A, Y53A, E55A) for 874F7-Hu1.

[0160] Figure 22 : Affinity of IL-33 mutant proteins (N61A, E62A, S63A, D65A, V67A, D68A, K70A) for 874F7-Hu1.

[0161] Figure 23 : Affinity of IL-33 mutant protein (L50A) for 874F7-Hu1.

[0162] Figure 24 : Position of key amino acid sites affecting 874F7-Hu1 binding in the IL-33 crystal 3D structure diagram. Specific implementation mode

[0163] After extensive and in-depth research and a large number of screenings, the present inventors obtained a series of anti-IL-33 humanized antibodies with excellent affinity. Specifically, the humanized antibodies of the present invention can block the binding of IL-33 and its receptor ST2, inhibit the secretion of IFNγ by IL-33-induced PBMC and NK cells, and inhibit the secretion of IL-5 and IL-13 by IL-33-induced basophilic leukemia cells KU812. The humanized antibodies of the present invention have obvious neutralizing activity in vivo. In a mouse animal experiment, a single treatment with the humanized antibody of the present invention can effectively reduce the increase in eosinophils in the peripheral blood of mice caused by human IL-33 stimulation. The antibodies of the present invention are expected to be used for the treatment of various IL-33-related diseases. On this basis, the present invention was completed.

[0164] Term

[0165] In the present invention, the terms "antibody (Ab for short)" and "immunoglobulin G (IgG for short)" are heterotetrameric glycoproteins with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also have regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region, and the heavy chain constant region is composed of three domains CH1, CH2, and CH3. One end of each light chain has a variable region (VL), and the other end has a constant region. The light chain constant region includes a domain CL; the constant region of the light chain pairs with the CH1 domain of the heavy chain constant region, and the variable region of the light chain pairs with the variable region of the heavy chain. The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC), etc. The heavy chain constant region includes IgG1, IgG2, IgG3, IgG4 subtypes; the light chain constant region includes κ (Kappa) or λ (Lambda). The heavy chain and light chain 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 covalently linked together by an inter-polypeptide disulfide bond formed between the hinge regions.

[0166] In the present invention, the terms "Fab" and "Fc" refer to the fact that papain can cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the heavy chain of the antibody and the VL and CL domains of the light chain. The Fc fragment, i.e., the fragment crystallizable (Fc), consists of the CH2 and CH3 domains of the antibody. The Fc fragment has no antigen-binding activity and is the site where the antibody interacts with effector molecules or cells.

[0167] In the present invention, the term "scFv" refers to a single chain antibody fragment (scFv), which is formed by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody usually through a linker peptide of 15 to 25 amino acids.

[0168] In the present invention, the term "variable" means that certain parts of the variable regions in an antibody are different in sequence, and it forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of the antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the variable regions of the heavy chain and the light chain. The more conserved parts in the variable regions are called framework regions (FRs). The variable regions of the natural heavy chain and light chain each contain four FR regions, which are generally in a β-sheet configuration and are connected by three CDRs forming connecting loops, and in some cases, a partial β-sheet structure can be formed. The CDRs in each chain are closely juxtaposed 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, Volume I, pages 647-669 (1991)).

[0169] As used herein, the term "framework region" (FR) refers to the amino acid sequences inserted between the CDRs, i.e., those portions of the variable regions of the light and heavy chains of immunoglobulins that are relatively conserved among different immunoglobulins in a single species. Each of the light and heavy chains of an immunoglobulin has four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can thus be designated (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain can thus be designated (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FRs of the present invention are human antibody FRs or derivatives thereof, which are substantially identical to the naturally occurring human antibody FRs, i.e., having a sequence identity of 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0170] Given the amino acid sequences of the CDRs, those skilled in the art can readily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0171] As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

[0172] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain to provide sufficient mobility for the domains of the light and heavy chains to fold into an exchanged dual variable domain immunoglobulin. In the present invention, the preferred linkers are Linker1 and Linker2, where Linker1 connects VH and VL of a single-chain antibody (scFv), and Linker2 is used to connect the scFv to the heavy chain of another antibody.

[0173] Examples of suitable linkers include single glycine (Gly) or serine (Ser) residues, and the identity and sequence of the amino acid residues in the linker can vary depending on the type of secondary structural elements to be achieved in the linker.

[0174] In the present invention, the antibodies of the present invention further include their conservative variants, which refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids in the amino acid sequence of the bispecific antibody of the present invention with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0175] Table A

[0176] Initial residue Representative substitution Preferred substitution 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

[0177] In the present invention, the terms "anti-", "bind", and "specifically bind" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. Generally, an antibody binds to the antigen with a equilibrium dissociation constant (KD) of less than about 10 -7 M, for example less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or smaller. In the present invention, 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. For example, the binding affinity between an antibody and an antigen is measured using Surface Plasmon Resonance (SPR) in a BIACORE instrument or the relative binding affinity between an antibody and an antigen is measured using ELISA.

[0178] In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitope of the present invention is the region in the antigen that is bound by the antibody.

[0179] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or their fragments or their fusion proteins. The polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0180] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods.

[0181] The present invention also relates to vectors containing the appropriate DNA sequences described above and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0182] Drug Composition and Application

[0183] The present invention also provides a composition. Preferably, 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. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the substances to be formulated and the disease to be treated. The formulated pharmaceutical composition 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), intracranial injection, or intracavitary injection. In the present invention, the term "pharmaceutical composition" means that the bispecific antibody of the present invention can form a pharmaceutical preparation composition together with a pharmaceutically acceptable carrier to exert its efficacy more stably. These preparations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, and at the same time protect the multi-functional 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 (such as 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. Such carriers include (but not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should match the administration route. The pharmaceutical composition of the present invention can be made into an injectable form, for example, prepared by a conventional method with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injectables and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, such as about 10 micrograms per kilogram of body weight per day - about 50 milligrams per kilogram of body weight. In addition, the bispecific antibody of the present invention can also be used together with other therapeutic agents.

[0184] When using the pharmaceutical composition, a safe and effective amount of the bispecific antibody or its immunoconjugate is administered to a mammal, where the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight - about 10 milligrams per kilogram of body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health status, which are all within the scope of the skills of a skilled physician.

[0185] Antibody-Drug Conjugate (ADC)

[0186] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0187] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, which is conjugated to the antibody, preferably by chemical conjugation. Among them, the effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule can be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0188] The antibody of the present invention and the effector molecule can be conjugated through a coupling agent. Examples of the coupling agent can be any one or several of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent is a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent using a carboxyl group can be any one or several of cis-aconitic anhydride-based coupling agents (such as cis-aconitic anhydride) and acylhydrazone-based coupling agents (the coupling site is acylhydrazone).

[0189] Certain residues on the antibody (such as Cys or Lys, etc.) are used to connect with various functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers), and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. The functional agent (such as a drug, a detection reagent, a stabilizer) is conjugated (covalently linked) to the antibody. The functional agent can be directly or indirectly connected to the antibody through a linker.

[0190] The antibody can be conjugated with a drug to form an antibody-drug conjugate (ADCs). Typically, the ADC contains a linker located between the drug and the antibody. The linker can be a degradable or non-degradable linker. The degradable linker typically degrades easily in the intracellular environment. For example, the linker degrades at the target site, so that the drug is released from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-based linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptide-based linker can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide bond linkers). The non-degradable linker typically releases the drug under the condition that the antibody is hydrolyzed by proteases.

[0191] Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive groups. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodo-, bromo- or chloro-substituted); haloesters (e.g., iodo-, bromo- or chloro-substituted); halomethyl ketones (e.g., iodo-, bromo- or chloro-substituted), benzyl halides (e.g., iodo-, bromo- or chloro-substituted); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-tris-(mercurimethyl) dioxane, and the counterion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonates. The linker can include, for example, a maleimide attached to the antibody through a thiol succinimide.

[0192] The drug can be any cytotoxic, cell growth inhibitory or immunosuppressive drug. In an embodiment, the linker attaches the antibody and the drug, and the drug has a functional group capable of bonding to the linker. For example, the drug can have an amino, carboxyl, thiol, hydroxyl, or keto group capable of bonding to the linker. In the case where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0193] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In the present invention, the drug-linker can be used to form an ADC in a single step. In other embodiments, bifunctional linker compounds can be used to form an ADC in a two-step 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 an ADC.

[0194] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently attached to the linker through a 1,3-dipolar cycloaddition between the azide and the alkyne. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazines and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other conjugation strategies, such as those described in Bioconjugate Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for the selective reaction of the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used either for the linker or for the drug.

[0195] The present invention also provides a method for preparing an ADC, which may further comprise: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0196] In certain embodiments, the method of the present invention comprises: combining an antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further comprises: combining the antibody-linker conjugate with a drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.

[0197] In some embodiments, the antibody-drug conjugate ADC is represented by the following formula:

[0198]

[0199] Wherein:

[0200] Ab is an antibody,

[0201] LU is a linker;

[0202] D is a drug;

[0203] And the subscript p is a value selected from 1 to 8.

[0204] The following examples are for further illustration of the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods, such as those for constructing vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in many publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold spring Harbor Laboratory Press.

[0205] The experimental materials and their sources used in the following examples and the preparation methods of the experimental reagents are specifically described as follows.

[0206] Experimental materials and reagents:

[0207] Balb / c mice: Purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0208] PBMC: Purchased from Ausabio Biotechnology Shanghai Co., Ltd., product number PB004-C.

[0209] Human IL-33-his: The Ser112-Thr270 of the IL-33 sequence (NCBI accession number NP_254274.1) was cloned into an E. coli expression vector for expression, and a 6×His tag was added to the C-terminus of IL-33 to facilitate purification through a Ni + affinity chromatography column, and its amino acid sequence is shown in SEQ ID NO: 53.

[0210] Cynomolgus monkey IL-33-his: Purchased from Sino Biological, product number 90912-CNAE.

[0211] IL-33-his-biotin protein: Purchased from Thermo Fisher, product number 20217, and the IL-33-his protein was biotinylated according to the instructions of the EZ-Link NHS-Biotin Reagent.

[0212] Rat-Anti-human IL-6: Purchased from BD Pharmingen, product number 554543.

[0213] biotin Rat Anti Human IL-6: Purchased from BD Pharmingen, product number 554546.

[0214] Mouse-Anti-human IFNγ: Purchased from BD Biosciences, catalog number 551221.

[0215] biotin mouse-anti-human IFNγ: Purchased from BD Biosciences, catalog number 554550.

[0216] Goat anti-mouse IgG secondary antibody: Purchased from Millipore, catalog number AP181P.

[0217] HRP-anti human IgG Fc secondary antibody: Purchased from Sigma, catalog number A0170.

[0218] HRP-labeled streptavidin secondary antibody: Purchased from BD Biosciences, catalog number 554066.

[0219] IL-6 kit: Invitrogen, catalog number 88-7066-77.

[0220] IL-8 kit: Invitrogen, catalog number BMS204-3TEN

[0221] IL-12: Purchased from Sino Biological, catalog number CT011-H08H.

[0222] ST2-Fc protein: The human ST2 sequence (NCBI accession number NP_003847.2) was fused with the Fc region sequence of human IgG1 and cloned into the eukaryotic expression vector PTT5. Fusion expression was carried out by transfecting HEK-293F cells, and then the expression supernatant was collected and purified through a Protein A affinity chromatography column. The amino acid sequence of ST2-Fc is shown in SEQ ID NO: 54.

[0223] TMB: Purchased from BD, catalog number 555214.

[0224] 1% Glutmine, 1% Sodium pyruvate, 1% MEM-NEAA (Minimum Essential Medium - Non-Essential Amino Acid Solution), 1% Penicillin-streptomycin, β-mercaptoethanol, 20% FBS (Fetal Bovine Serum): All purchased from Gibco.

[0225] SA protein: Streptavidin, purchased from Sigma, catalog number 85878-1MG.

[0226] SFM medium: Purchased from life technologies, product number 12045-076.

[0227] RPMI 1640 complete medium: Purchased from Gibco.

[0228] Experimental instruments:

[0229] Electrofusion instrument: Purchased from BTX.

[0230] Microplate reader: Purchased from Molecular Devices, model SpectraMax 190.

[0231] The antibody sequences of the present invention are shown in the following table:

[0232]

[0233]

[0234]

[0235] Example 1 Immunization of animals with antigen and preparation and screening of hybridomas

[0236] Step 1: Immunize mice with antigen

[0237] Balb / c mice were immunized intraperitoneally with the prokaryotic recombinant expressed human IL-33-his protein routinely. On the first day, after the soluble human IL-33-his protein was fully mixed with Freund's complete adjuvant or water-soluble adjuvant (quick antibody), Balb / c mice were injected intraperitoneally (50 μg / mouse of human IL-33-his). On the fourteenth day, after the soluble human IL-33-his protein was fully mixed with Freund's incomplete adjuvant or water-soluble adjuvant (quick antibody), Balb / c mice were boosted intraperitoneally (50 μg / mouse of human IL-33-his). On the thirty-sixth day, the animals were boosted with the soluble human IL-33-his protein in the same way as last time (50 μg / mouse of human IL-33-his). Three weeks later, the mice were injected intraperitoneally with human IL-33-his for stimulation. 3 - 4 days later, the spleens of the mice were taken for fusion experiments.

[0238] Step 2: Preparation and screening of hybridomas

[0239] Three to four days after the last boost immunization of the mice, using a conventional hybridoma technology protocol, mouse spleen cells and mouse myeloma cells SP2 / 0 were electrofused using an electrofusion apparatus. The fused cells were evenly suspended in complete medium, which was prepared by mixing RPMI1640 and DMEM F12 media at a ratio of 1:1 and then adding 1% Glutmine, 1% Sodium pyruvate, 1% MEM-NEAA, 1% Penicillin-streptomycin, 50 μM β-mercaptoethanol, and 20% FBS. At a density of 10 5 cells / 100 μl / well, the cells were seeded into a total of 36 96-well culture plates and cultured overnight. The next day, 100 μl of complete medium containing 2×HAT was added to each well, making the culture medium in the 96-well plates 200 μl / well (containing 1×HAT). Seven to twelve days later, the supernatant was harvested, and hybridoma wells positive for human IL-33-his binding activity were screened by indirect enzyme-linked immunosorbent assay (ELISA).

[0240] Among them, the method for screening hybridoma wells positive for human IL-33-his binding activity by indirect enzyme-linked immunosorbent assay is as follows: The recombinant human IL-33-his protein was diluted to 1 μg / ml with coating buffer (50 mM carbonate coating buffer, pH 9.6), and 100 μl / well was added to the enzyme-linked immunosorbent assay plate and coated overnight at 4°C. The plate was washed 3 times with PBST, 200 μl / well of blocking solution (2% BSA-PBS) was added, and after incubation at 37°C for 1 h, the plate was washed once with PBST and set aside. The harvested hybridoma supernatant was sequentially added to the blocked enzyme-linked immunosorbent assay plate, 100 μl / well, and incubated at 37°C for 1 h. The plate was washed 3 times with PBST, HRP-labeled goat anti-mouse IgG secondary antibody was added, and incubated at 37°C for 30 min; after washing the plate 5 times with PBST, the residual liquid drops were blotted dry as much as possible on absorbent paper, 100 μl of TMB was added to each well, and the plate was incubated in the dark at room temperature (20 ± 5°C) for 5 min; 50 μl of 2M H2SO4 stop solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm using an enzyme-linked immunosorbent assay reader to analyze the binding ability of the antibody to be tested to the target antigen human IL-33-his.

[0241] The 10 hybridoma cell lines obtained by amplification and screening in serum-containing complete medium were centrifuged and the medium was changed to serum-free SFM medium to a cell density of 1 - 2×10 7 / ml, cultured for 2 weeks under the conditions of 5% CO2 and 37 °C, centrifuged to obtain the culture supernatant, and purified by Protein G affinity chromatography to obtain 10 murine anti-human IL-33 monoclonal antibodies. They were named 874F7, 871G1, 864F3, 887B9, 858D5, 868H10, 604A8, 604A12, 646F8, and 651H2 respectively.

[0242] Example 2 Determination of the binding activity of murine antibodies to human IL-33 by ELISA

[0243] The indirect enzyme-linked immunosorbent assay was used to determine the binding ability of murine antibodies to human IL-33. The specific method was as follows: Biotin-avidin (SA) was pre-coated and diluted to 2 μg / ml with coating buffer (50 mM carbonate coating buffer, pH 9.6), then coated on the plate at 4 °C overnight; then blocked with 5% skim milk powder at 37 °C for 2 hours. Stored at -80 °C for later use. The biotinylated recombinant human IL-33-his protein was diluted to 0.5 μg / ml with coating buffer (50 mM carbonate coating buffer, pH 9.6), and 100 μl / well was added to the pre-coated SA plate, incubated at 37 °C for 0.5 hour. The plate was washed 3 times with PBST. The test antibodies serially diluted with 1% BSA were added to the blocked enzyme-linked immunosorbent assay plate in turn, 100 μl / well, and incubated at 37 °C for 1 h. The plate was washed 3 times with PBST, and the secondary antibody of HRP-labeled goat anti-mouse IgG was added, incubated at 37 °C for 30 min; after washing the plate 3 times with PBST, the residual liquid drops were patted dry as much as possible on the absorbent paper, 100 μl of TMB was added to each well, and incubated at room temperature (20 ± 5 °C) in the dark for 5 min; 50 μl of 2M H2SO4 termination solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm with an enzyme-linked immunosorbent assay reader to analyze the binding ability of the test antibody to the target antigen human IL-33-his.

[0244] The results were as Figure 1A 、 1B , and the indicated antibodies all had good binding activity. The EC 50 values of the antibodies 874F7, 871G1, 864F3, 887B9, 858D5, and 868H10 were 0.11 nM, 0.14 nM, 0.27 nM, 0.07 nM, 0.18 nM, and 0.36 nM respectively. The EC 50 values of the antibodies 604A8, 604A12, 646F8, and 651H2 were 0.11 nM, 0.16 nM, 0.10 nM, and 0.13 nM respectively.

[0245] Example 3 Determination of the activity of murine antibodies blocking the binding of ST2 to IL-33

[0246] ST2 is the only receptor of IL-33 discovered so far. As a cytokine, IL-33 exerts its physiological functions through the ST2 signaling pathway, and the blocking activity of antibodies can reflect their neutralizing activity. The specific method is as follows: Coat the plate with 2 μg / ml ST2 PBS solution and incubate overnight at 4°C; Wash 3 times with PBST, then block with 5% skim milk powder at 37°C for 2 hours; Wash 3 times with PBST for later use; Mix 20 ng / ml IL-33-his-biotin antigen diluted with 1% BSA and the antibody to be tested diluted with 1% BSA in a gradient at a 1:1 equal volume ratio and incubate at 37°C for hours; Add this mixture to the pre-coated ST2 plate and incubate at 37°C for 1 hour; Wash 3 times with PBST; Add HRP-labeled streptavidin secondary antibody at a ratio of 1:8000 and incubate at 37°C for 0.5 hour; After washing the plate 3 times with PBST, pat dry the residual liquid droplets as much as possible on the absorbent paper, add 100 μl of TMB to each well, and place in the dark at room temperature (20 ± 5°C) for 5 min; Add 50 μl of 2 M H2SO4 termination solution to each well to terminate the substrate reaction, and read the OD value at 450 nm with an enzyme-linked immunosorbent assay reader.

[0247] The results are shown in Figure 2A 、 2B , Figure 2A The antibodies 874F7, 871G1, 864F3, 887B9, 858D5, 868H10 shown have good blocking activities, and the IC 50 values are 0.90 nM, 0.74 nM, 0.23 nM, 0.25 nM, 0.16 nM, and 0.27 nM respectively. Figure 2B The antibodies 604A8, 604A12, 646F8, 651H2 shown have no or very weak blocking activities.

[0248] Example 4 Inhibitory Effect of Murine Antibodies on IL-6 Expression Induced by IL-33 in HUVEC Cells

[0249] The biological activity of the anti-IL-33 antibody was determined using HUVEC cells. The specific method was as follows: HUVEC cells were cultured in a T75 culture flask. The HUVEC cells were passaged to the fifth generation. The HUVEC cells in the T75 culture flask were washed with 1×PBS, then digested with 1 ml of trypsin for 10 min. After the cells detached, the digestion was terminated with 10 ml of medium. The cells were counted and plated at 100 μl / well, 6000 cells / well. The remaining cells were continued to be cultured and cryopreserved in the T75 culture flask. IL-33-his was diluted to 20 ng / ml with medium. The antibody to be tested was serially diluted 8-fold from 40 μg / ml. Four blank wells were set up. After dilution, 50 μl of the antigen and the antibody were each added to the cell plate and mixed well. That is, the final concentration of IL-33-his was 5 ng / ml. After mixing, it was incubated at 37 °C for 12 h to 18 h. The supernatant was taken to measure the concentration of IL-6. The measurement method was detailed in the IL-6 kit instruction manual. The IL-6 inhibition rate was calculated according to the following formula and analyzed by fitting with GraphPad Prism 6 software:

[0250] Inhibition rate = (ODAgIL-33 - ODadministered) / (ODAgIL-33 - ODblank) × 100%.

[0251] The results were as Figure 3A 、 3B shown in Figure 3C, and the IC50 values of the inhibitory activities of 874F7, 871G1, 864F3, 887B9, 858D5, 868H10, 604A8, 604A12, 651H2 were 50 0.65 nM, 1.66 nM, 1.52 nM, 4.53 nM, 12.25 nM, 9.35 nM, 0.08 nM, 0.13 nM, 0.04 nM, respectively. In addition, the maximum inhibition rates of antibodies 604A8, 604A12, and 651H2 were relatively poor.

[0252] Example 5 Inhibitory effect of murine antibodies on IL-33-induced IL-8 expression in HUVEC cells

[0253] The biological activity of the anti-IL-33 antibody was determined using HUVEC cells. The specific method is as follows: HUVEC cells were cultured in a T75 culture flask. The HUVEC cells were passaged to the fifth generation. The HUVEC cells in the T75 culture flask were washed with 1×PBS, then digested with 1 ml of trypsin for 10 min. After the cells detached, the digestion was terminated with 10 ml of medium. The cells were counted and plated at 100 μl / well, 6,000 cells / well. The remaining cells in the T75 culture flask were continued to be cultured and cryopreserved. IL-33-his was diluted to 20 ng / ml with medium. The antibody to be tested was serially diluted 8-fold from 40 μg / ml. Four wells were used as blanks. After dilution, 50 μl each of the antigen and antibody were added to the cell plate and mixed well. That is, the final concentration of IL-33-his was 5 ng / ml. After mixing, the cells were incubated at 37 °C for 12 h to 18 h. The supernatant was taken to measure the concentration of IL-8. The measurement method is detailed in the IL-8 kit instruction manual. The IL-8 inhibition rate was calculated according to the following formula and fitted and analyzed using GraphPad Prism 6 software:

[0254] Inhibition rate = (ODAgIL-33 - OD administered) / (ODAgIL-33 - OD blank) × 100%.

[0255] The results are shown in Figure 4 , the activities of antibodies 858D5 and 868H10 were very weak, and the IC 50 values of antibodies 874F7, 871G1, 864F3, and 887B9 were 0.35 nM, 0.16 nM, 1.46 nM, and 8.90 nM, respectively.

[0256] Example 6 Cross-reactivity determination of murine antibodies with cynomolgus monkey IL-33 antigen

[0257] Indirect enzyme-linked immunosorbent assay was used to determine the binding ability of murine antibodies to cynomolgus monkey IL-33. The specific method was as follows: Biotin-avidin (SA) was pre-coated on the plate by diluting it to 2 μg / ml with coating buffer (50 mM carbonate coating buffer, pH 9.6) at 4 °C overnight; then blocked with 5% skim milk at 37 °C for 2 hours. Stored at -80 °C for later use. Biotinylated recombinant cynomolgus monkey IL-33-his protein was diluted to 1 μg / ml with coating buffer (50 mM carbonate coating buffer, pH 9.6), and 100 μl per well was added to the pre-coated SA plate at 37 °C for 0.5 hour. The plate was washed 3 times with PBST. The test antibodies serially diluted with 1% BSA were added to the blocked enzyme-linked immunosorbent assay plate in turn, 100 μl per well, and incubated at 37 °C for 1 h. The plate was washed 3 times with PBST, and HRP-labeled goat anti-mouse IgG secondary antibody was added and incubated at 37 °C for 30 min; after washing the plate 3 times with PBST, the residual liquid drops were patted dry as much as possible on the absorbent paper, 100 μl of TMB was added to each well, and incubated at room temperature (20 ± 5 °C) in the dark for 5 min; 50 μl of 2M H2SO4 termination solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm with an enzyme-linked immunosorbent assay reader to analyze the binding ability of the test antibody to the target antigen cynomolgus monkey IL-33-his.

[0258] The results are shown in Figure 5 , and all the indicated antibodies could bind well to cynomolgus monkey IL-33 protein. The EC 50 values of antibodies 874F7, 871G1, 864F3, 887B9, 858D5, 868H10, 604A8, 604A12, 646F8, and 651H2 were 0.03 nM, 0.04 nM, 0.06 nM, 0.05 nM, 0.02 nM, 0.05 nM, 0.06 nM, 0.06 nM, 0.03 nM, and 0.06 nM, respectively.

[0259] Example 7 Evaluation of in vivo neutralizing activity of murine antibodies

[0260] Intravenous administration of antigen IL-33 to animals can cause changes in the body's inflammatory response, proliferation of spleen cells, and thus an increase in spleen weight. This method can be used to evaluate the activity of antibody drugs in neutralizing 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 first group was the normal control group; the second group was the IL-33-his antigen challenge group, and starting from the second day after grouping, 0.4 μg / animal of IL-33 antigen was intraperitoneally injected every day for 6 consecutive days; the third group was the 864F3 treatment group. On the day of grouping, 864F3 antibody was intraperitoneally injected at 5 mg / kg, and starting from the second day after grouping, 0.4 μg / animal of IL-33 antigen was intraperitoneally injected every day for 6 consecutive days; the fourth group was the 871G1 treatment group. On the day of grouping, 871G1 antibody was intraperitoneally injected at 5 mg / kg, and starting from the second day after grouping, 0.4 μg / animal of IL-33-his antigen was intraperitoneally injected every day for 6 consecutive days; the fifth group was the 874F7 treatment group. On the day of grouping, 874F7 antibody was intraperitoneally injected at 5 mg / kg, and starting from the second day after grouping, 0.4 μg / animal of IL-33 antigen was intraperitoneally injected every day for 6 consecutive days. On the 7th day after grouping, the animals were weighed, and after euthanasia, the spleens were removed and weighed.

[0261] The experimental results are shown in Figure 6 , the average spleen weight of the PBS control group was 86.9 ± 9.4 mg, while the weight gain of the group administered with antigen Ag-IL-33 was significantly increased to 195.7 ± 26.9 mg. The average spleen weights of the groups administered with 864F3, 871G1, and 874F7 antibodies were 106.3 ± 24.5 mg, 77.7 ± 17.5 mg, and 89.9 ± 14.7 mg respectively, all showing significant neutralizing activity.

[0262] Example 8 Preparation of Chimeric Antibodies

[0263] In this example, the heavy chain variable regions and light chain variable regions of hybridomas 864F3, 874F7, and 871G1 were obtained through relevant molecular biology methods, and chimeric antibodies were further constructed.

[0264] RNA of the three hybridoma cells 864F3, 874F7, and 871G1 was extracted by Trizol and reverse transcribed into cDNA. Subsequently, using cDNA as a template, PCR was performed with degenerate primers for the heavy chain and light chain of murine antibodies (《Antibody Engineering》Volume 1, Edited by Roland Kontermann and Stefan Dübel, the sequences of the combined primers are from page 323). The obtained PCR products were sequenced and analyzed through the Kabat database to determine that the obtained sequences were the variable region sequences of murine antibodies.

[0265] The relevant sequence information is as follows:

[0266] 864F3 has two heavy chain variable region gene sequences, both with a full length of 363 bp, each encoding 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 respectively; the light chain variable region gene sequence of 864F3 has a full length of 321 bp, 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: 6.

[0267] The full length of the heavy chain variable region gene sequence of 874F7 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; the full length of the light chain variable region gene sequence of 874F7 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.

[0268] The full length of the heavy chain variable region gene sequence of 871G1 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; the full length of the light chain variable region gene sequence of 871G1 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.

[0269] The three preferred murine anti-IL-33 antibodies have relatively high homology in their 3 H-CDRs and 3 L-CDRs, especially they all have the same L-CDR2 (SEQ ID No: 22).

[0270] The obtained heavy chain variable region sequences of each hybridoma were spliced with the human IgG4 constant region (including the S228P mutation) (amino acid sequence shown in SEQ ID NO: 13), and the light chain variable region sequences were spliced with the human kappa chain constant region (amino acid sequence shown in SEQ ID NO: 14). The heavy and light chains of each chimeric antibody were respectively constructed into the pcDNA3.4 expression vector, transfected into HEK-293F cells, and each chimeric antibody was obtained by Protein A purification. The molecular weights of the expressed antibodies were determined to be about 150 kD by SDS-PAGE electrophoresis and SEC-HPLC, the antibody purity > 95%, quantified, aliquoted, and stored at -80 °C for later use.

[0271] Example 9 Determination of the binding activity of each chimeric antibody to human IL-33 by ELISA

[0272] Dilute the SA protein to 2 μg / mL, coat the ELISA plate, 100 μL per well, coat overnight at 4°C, wash 3 times with PBST (PBS containing 0.05% Tween 20), prepare 2% BSA with PBS, 200 μL per well, block at room temperature for 2 h, after washing 2 times with PBST, dilute the IL-33-his-biotin protein to 1 μg / mL with 1% BSA prepared with PBST, add to the ELISA plate at 100 μL per well, incubate at room temperature for 1 h, dilute each monoclonal antibody with 1% BSA prepared with PBST in 3-fold gradients, the highest concentration is 10 μg / mL, dilute 12 gradients, add to the ELISA wells, 100 μL per well, incubate at room temperature for 1 h, make 2 duplicate wells in parallel for each sample, wash 3 times with PBST, dilute the HRP-anti human IgG Fc secondary antibody with 1% BSA prepared with PBST in an appropriate ratio, add to the ELISA wells, 100 μL per well, incubate at room temperature for 1 h, after washing 3 times with PBST, add the TMB chromogenic solution, 100 μL per well, develop color until the expected color, terminate the color reaction with 2M H2SO4, 70 μL per well, make the reaction solutions oscillate evenly and measure the OD450nm with an enzyme-linked immunosorbent assay reader, analyze the data, and calculate the EC 50 。

[0273] The experimental results are as Figure 7 shown. The EC 50 values of the chimeric antibodies 864F3-ch2, 864F3-ch1, 871G1-ch, and 874F7-ch are 0.173 nM, 0.127 nM, 0.178 nM, and 0.144 nM respectively, indicating that each chimeric antibody has good affinity for IL-33.

[0274] Example 10 Determination of the Activity of the Chimeric Antibody in Blocking the Binding of ST2 to IL-33

[0275] Dilute the ST2-Fc protein with ELISA coating buffer to 1 μg / mL, coat the ELISA plate, 100 μL per well, place it in a wet box, at 4 °C, coat for 16 h, wash the ELISA plate 3 times with PBST, then use 2% BSA prepared with PBS, 200 μL per well, block at room temperature for 2 h, wash 1 time with PBST, pat dry, remove the excess blocking solution, dilute each monoclonal antibody with 1% BSA PBST at 3-fold gradients, the highest concentration is 40 μg / mL, dilute for 11 gradients, mix the diluted antibody with 10 ng / ml IL-33-his-biotin protein at a ratio of 1:1, after mixing evenly, add to the ELISA wells, 100 μL per well, incubate at room temperature for 1 h, make 2 replicates in parallel for each concentration (the final concentration of the highest antibody concentration is 20 μg / mL, and the final concentration of IL-33-his-biotin is 5 ng / mL), wash away the unbound or non-specifically bound primary antibody, according to the requirements of the antibody instruction manual, dilute the HRP-labeled streptavidin secondary antibody with antibody diluent to an appropriate concentration, add to the ELISA plate, 100 μL per well, incubate at room temperature for 1 h, wash 5 times with PBST, and pat dry the ELISA plate on absorbent paper to remove the excess liquid, add TMB chromogenic solution, 100 μL per well, develop color to an appropriate depth, add 2M H2SO4, 70 μL per well, to terminate the color development, and measure its absorbance at a wavelength of 450 nm in a multi-functional microplate reader, and analyze the data.

[0276] The experimental results are as Figure 8 shown, the IC 50 values of the chimeric antibodies 874F7-ch, 871G1-ch, 864F3-ch1, and 864F3-ch2 are 0.634 nM, 0.853 nM, 45.245 nM, and 0.580 nM respectively, indicating that each chimeric antibody is an IL-33 blocking antibody and can effectively block the binding of IL-33 to its receptor ST2, and 874F7-ch, 871G1-ch, and 864F3-ch2 are relatively superior.

[0277] Preparation of humanized antibody in Example 11

[0278] Analyze the amino acid sequences of the light chain variable region and heavy chain variable region of each candidate murine antibody, and determine the 3 antigen complementary determining regions (CDRs) and 4 framework regions (FRs) of the murine antibody according to the Kabat rules. Among them, the amino acid sequence of the heavy chain complementary determining region of 864F3 is

[0279] HCDR1: NYGVH (SEQ ID NO: 15),

[0280] HCDR2: VIRAGGSSDYNSALMS (SEQ ID NO: 16),

[0281] HCDR3: DHYFSNSYGGSPY (SEQ ID NO: 17) or

[0282] HCDR1: KYGVH (SEQ ID NO: 18),

[0283] HCDR2: VLRAGGTISYNSALMS (SEQ ID NO: 19),

[0284] HCDR3: DHYYYSSFGGFAS (SEQ ID NO: 20),

[0285] The amino acid sequence of the light chain complementarity determining region is

[0286] LCDR1: LASQTIATWLA (SEQ ID NO: 21),

[0287] LCDR2: AATRLAD (SEQ ID NO: 22) and

[0288] LCDR3: QQLYNTPYT (SEQ ID NO: 23).

[0289] The amino acid sequence of the heavy chain complementarity determining region of 874F7 is

[0290] HCDR1: KYGVH (SEQ ID NO: 18),

[0291] HCDR2: VLRAGGSTGYNSALMS (SEQ ID NO: 24),

[0292] HCDR3: DHYYYSSYGGFVY (SEQ ID NO: 25),

[0293] The amino acid sequence of the light chain complementarity determining region is

[0294] LCDR1: LASQTIGAWLA (SEQ ID NO: 26),

[0295] LCDR2: AATRLAD (SEQ ID NO: 22) and

[0296] LCDR3: QQLDSSPYT (SEQ ID NO: 27).

[0297] The amino acid sequence of the heavy chain complementarity determining region of 871G1 is

[0298] HCDR1: KYGVH (SEQ ID NO: 18),

[0299] HCDR2: VLRAGGTISYNSALMS (SEQ ID NO: 19),

[0300] HCDR3: DHYYYSSFGGFAS (SEQ ID NO: 20),

[0301] The amino acid sequences of the light chain complementarity-determining regions are

[0302] LCDR1: LASQTIGAWLA (SEQ ID NO: 26),

[0303] LCDR2: AATRLAD (SEQ ID NO: 22) and

[0304] LCDR3: QQLNSTPYT (SEQ ID NO: 28).

[0305] The CDR sequences of the murine antibodies are shown in Table 1a as follows:

[0306]

[0307] Humanized templates that best match the non-FR regions of the above murine antibodies were selected from the Germline database. Then, the CDR regions of the murine antibodies were transplanted onto the selected humanized templates to replace the CDR regions of the human templates. The heavy chain variable regions were recombined with the human IgG4 constant region (including the S228P mutation), and the light chain variable regions were recombined with the human kappa chain constant region. At the same time, based on the three-dimensional structure of the antibody, back mutations were performed on the buried residues, the residues that directly interact with the CDR regions, and the residues that have an important impact on the conformations of the VL and VH of each antibody. Finally, multiple humanized antibodies were obtained. The heavy and light chain variable regions and sequences corresponding to each humanized antibody and chimeric antibody are shown in Table 1b. The heavy and light chains of each humanized antibody were constructed into the pcDNA3.4 expression vector and transfected into HEK-293F cells. Each humanized antibody was obtained by Protein A purification, and the molecular weight of each antibody was determined to be correct and the purity > 95% by SDS-PAGE electrophoresis and SEC-HPLC.

[0308] Table 1b: Variable region sequence list of each humanized antibody

[0309]

[0310]

[0311] Example 12: Determination of the binding activity of each humanized antibody to human IL-33 by ELISA

[0312] The binding affinity of each of the above humanized antibodies to human IL-33 was determined by ELISA, and the relevant experimental methods were referred to Example 9.

[0313] The experimental results are as Figure 9A - 9D shown. The EC 50 values of chimeric antibody 864F3-ch2 and humanized antibodies 864F3-HuG, 864F3-Hu1, and 864F3-Hu2 were 0.169 nM, 0.760 nM, 0.249 nM, and 0.181 nM, respectively. Compared with chimeric antibody 864F3-ch2, the humanized antibodies 864F3-HuG, 864F3-Hu1, and 864F3-Hu2 had more affinity loss for human IL-33. The EC 50 values of chimeric antibodies 864F3-ch2 and 864F3-Hu3, 864F3-Hu4, and 864F3-Hu5 were 0.124 nM, 0.138 nM, 0.135 nM, and 0.146 nM, respectively. Compared with chimeric antibody 864F3-ch2, the affinities of 864F3-Hu3, 864F3-Hu4, and 864F3-Hu5 for human IL-33 were almost unchanged.

[0314] The EC 50 values of chimeric antibody 874F7-ch and humanized antibodies 874F7-Hμg, 874F7-Hu1, and 874F7-Hu2 were 0.177 nM, 0.170 nM, 0.129 nM, and 0.136 nM, respectively. Compared with chimeric antibody 874F7-ch, the humanized antibodies 874F7-Hu1 and 874F7-Hu2 did not show affinity loss for human IL-33.

[0315] The EC 50 values of chimeric antibody 871G1-ch and humanized antibodies 871G1-HuG, 871G1-Hu1, and 871G1-Hu2 were 0.255 nM, 0.187 nM, 0.191 nM, and 0.176 nM, respectively. Compared with chimeric antibody 871G1-ch, the humanized antibodies 871G1-Hu1, 871G1-Hu2, and 871G1-HuG did not show affinity loss for human IL-33.

[0316] Example 13 Determination of the Activity of Humanized Antibodies in Blocking the Binding of ST2 to IL-33

[0317] The blocking effect of each of the above humanized antibodies on the binding of IL-33 to its receptor ST2 was determined by ELISA, and the relevant experimental methods were referred to Example 10.

[0318] The experimental results are as Figure 10As shown, the IC 50 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 were 0.512 nM, 0.473 nM, 0.404 nM, 0.361 nM, 0.451 nM, and 0.350 nM, respectively. This indicates that each humanized antibody retained the blocking effect on the binding of IL-33 to ST2.

[0319] Example 14 Determination of the Binding Kinetics of Humanized Monoclonal Antibodies to IL-33

[0320] Capture each antibody to be tested 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 is 2 μg / mL, contact time is 75 s, flow rate is 10 μL / min, and regeneration contact time is 30 s. Dilute the IL-33-his antigen using HBS-EP pH 7.4 buffer (purchased from GE Healthcare, product number BR-1006-69), with the highest concentration being 50 nM, diluted 2-fold to 0.39 nM, set up replicate wells and a 0-concentration point, use 6 M guanidine hydrochloride solution as the regeneration buffer, and inject samples on the Biacore 8K according to the following parameters: binding time is 180 s, dissociation time is 900 s, flow rate is 30 μL / min, and regeneration contact time is 30 s. After the run is completed, use the Biacore 8K Evaluation Software to analyze the data according to the "1:1 binding kinetics model" to obtain the binding kinetics parameters of each antibody to IL-33.

[0321] The results are shown in Table 2. The association constants (ka), dissociation constants (kd), and equilibrium dissociation constants (KD) of each humanized antibody for IL-33 were at the same level.

[0322] Table 2: Binding Kinetics Parameters of Each Humanized Antibody to IL-33

[0323] Sample 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

[0324] Example 15 Inhibitory Effect of Humanized Antibodies on IL-6 Expression Induced by IL-33 in HUVEC Cells

[0325] In this example, the activity of each humanized antibody was evaluated by measuring the inhibitory effect of each humanized antibody on the expression of IL-6 in HUVEC cells induced by IL-33-his protein. The specific experimental steps are as follows: After digesting and counting the HUVEC cells in a T75 culture flask, plate them in complete medium, 100 μL / well, 6000 cells / well, and culture them in an incubator at 37°C. After 2 h, start dosing after the cells adhere; Dilute the antibody to be tested with complete medium, with the highest concentration being 200 nM, and dilute it in 9 gradients of 5-fold dilution. Set positive and negative blank controls; Mix the diluted antibody with 20 ng / mL IL-33-his in a volume ratio of 1:1 (the final highest concentration of the antibody after adding to the cells is 50 nM, and the final concentration of IL-33-his is 5 ng / mL). Incubate at room temperature for 30 min; Take 100 μL of the antibody / IL-33-his mixture and add it to the cell solution, mix gently, and after acting in a 37°C CO2 incubator for 20 h, centrifuge to collect the supernatant and store it at -80°C for IL-6 determination.

[0326] Dilute Rat-Anti-human IL-6 protein with ELISA coating buffer to 2.5 μg / mL, coat the ELISA plate, 100 μL / well, place it in a wet box, and coat at 4°C for 16 h; Wash the ELISA plate three times with PBST to remove unbound protein, and pat the ELISA plate dry on absorbent paper to remove excess liquid. Then, block it with 2% BSA prepared with PBS, 200 μL / well, at room temperature for 1 - 2 h; Dilute the IL-6 standard product with 1% BSA prepared with PBS in 3-fold gradients, with the highest concentration being 30 ng / mL. After mixing evenly, add it to the ELISA wells, 100 μL / well, add 100 μL / well of the above cell supernatant to the ELISA wells, incubate at room temperature for 1 h, and make 2 replicates for each sample in the standard curve; Wash away the unbound or non-specifically bound primary antibody, dilute biotin RatAnti Human IL-6 with antibody diluent at a ratio of 1:1000, mix evenly and add it to the ELISA wells, 100 μL / well, and incubate at room temperature for 1 h; Wash away the unbound or non-specifically bound antibody, dilute the HRP-labeled streptavidin secondary antibody to an appropriate concentration, add it to the ELISA plate, 100 μL / well, and incubate at room temperature for 1 h; Wash 5 times with PBST, pat the ELISA plate dry on absorbent paper to remove excess liquid, add TMB chromogenic solution, develop color to an appropriate depth, add 2M H2SO4, 50 μL / well, to terminate the color development, and measure its absorbance at a wavelength of 450 nm in a multi-functional microplate reader to analyze the data.

[0327] The experimental results are as Figure 11As shown, the IC of the inhibitory effects of the humanized antibodies 864F3-Hu4, 864F3-Hu5, 871G1-Hu1, 871G1-Hu2, 874F7-Hu1, and 874F7-Hu2 on the IL-6 expression induced by IL-33 in HUVEC cells 50 values were 0.073 nM, 0.147 nM, 0.210 nM, 0.175 nM, 0.051 nM, and 0.050 nM, respectively. This indicates that each humanized antibody can effectively inhibit the secretion of IL-6 by IL-33-his protein in HUVEC cells, and 874F7-Hu1 and 874F7-Hu2 have relatively better effects.

[0328] Example 16 Inhibitory Effect of Humanized Antibodies on IFNγ Secretion Induced by IL-33 in PBMC

[0329] In this example, the activities of the humanized antibodies were evaluated by measuring the inhibitory effects of the humanized antibodies on the IFNγ secretion induced by IL-33-his protein in PBMC. The specific experimental steps were as follows: Fresh PBMC was centrifuged and counted, washed once with PBS, and diluted to 4×10 6 cells / mL with RPMI1640 complete medium; PBMC cells were added to a 96-well U-bottom cell culture plate, 50 μL per well (2×10 5 cells / well), and placed in a 37°C incubator to recover; the antibody was diluted with RPMI1640 complete medium, with the highest concentration of 200 nM (final concentration of 50 nM), and diluted in a 3-fold gradient. After dilution, an equal volume of 40 ng / mL IL-33-his (final concentration of 10 ng / mL) was added to each well, and incubated at 37°C for 30 min; 50 μL of IL-12 diluted with 40 ng / mL complete medium was added to the above PBMC cell culture plate, and then 100 μL of the antibody and IL-33-his mixture was added. After mixing, it was placed in a 37°C cell culture incubator for 24 h; the cell culture plate was centrifuged at 500 g for 5 min, and 180 μL of the supernatant was aspirated from each well for detection.

[0330] Mouse-Anti-human IFNγ antibody (purchased from BD Biosciences, catalog number 551221) was diluted to 1 μg / mL with ELISA coating solution, coated on ELISA plate, 100 μL / well, placed in a humidified box, 4°C, coated for 16 hours; washed ELISA plate three times with PBST, and patted the ELISA plate dry on absorbent paper to remove excess liquid, then blocked with 2% BSA prepared in PBS, 200 μL / well, at room temperature for 1-2 hours; washed once with PBST to remove excess blocking solution, and patted the ELISA plate dry to remove excess liquid; diluted IFNγ standard, the highest concentration was 250 ng / mL, 1 / 2 dilution 12 gradients, added to ELISA wells, 100 μL / well, and each sample was made in parallel with 2 replicate wells; added 100 μL of the above-mentioned cell supernatant to be tested to the ELISA plate, incubated at room temperature for 1 hour; washed three times with PBST, added biotin diluted 1:1000 with 1% BSA PBST mouse-anti-human IFNγ (purchased from BD, catalog number 554550), 100 μL / well, incubated at room temperature for 1 hour; unbound or non-specifically bound antibodies were washed away, and HRP-labeled Streptavidin (purchased from BD, catalog number 554066) was diluted to an appropriate concentration with 1% BSA PBST according to the antibody instructions, added to the ELISA plate, 100 μL / well, and incubated at room temperature for 1 hour; washed five times with PBST, and the ELISA plate was patted dry on absorbent paper to remove excess liquid, TMB color development solution was added, 100 μL / well, color developed to an appropriate depth, 2MH2SO4, 50 μL / well was added to stop color development, and its absorbance was measured at a wavelength of 450 nm in a multifunctional microplate reader, and the data was analyzed.

[0331] The experimental results are as follows Figure 12 As shown, the inhibitory effects of humanized antibodies 864F3-Hu4, 864F3-Hu5, 871G1-Hu1, 871G1-Hu2, 874F7-Hu1, and 874F7-Hu2 on IL-33-induced IFNγ secretion from PBMCs were 50 The values ​​were 1.224nM, 0.839nM, 1.395nM, 1.061nM, 0.850nM, and 1.736nM, respectively. This indicates that all humanized antibodies can effectively inhibit IL-33-his protein from inducing IFNγ secretion from human PBMC, among which 874F7-Hu2 has relatively poor activity.

[0332] Example 17 Inhibitory effect of humanized antibodies on IL-33-induced NK cell secretion of IFNγ

[0333] After centrifuging and counting fresh PBMCs, NK cells were isolated according to the instructions of the Nk Cell Isolation Kit human (purchased from Miltenyi, catalog number 130-092-657). The NK cells were washed twice with PBS, counted, and diluted to 0.8×10 6 cells / mL with 1640 + 10% FBS + 1% Glutamax medium, and 96-well cell culture plates were seeded with 50 μL of the cell suspension per well. Each antibody was diluted with 1640 + 10% FBS + 1% Glutamax medium, and the final highest working concentration was 100 nM, diluted in a 1 / 4 gradient. After dilution, IL-33-his with a final concentration of 10 ng / mL was added to each well and incubated in a 37°C cell culture incubator for 30 min. Subsequently, IL-12 with a final concentration of 2 ng / mL diluted in complete medium was added to the 96-well cell culture plates containing NK cells. Finally, 100 μL of the mixture of the above antibody and IL-33 was added, and after incubation in a 37°C cell culture incubator for 24 h, the cell culture supernatant was collected to measure the secretion level of IFNγ.

[0334] The detection method of IFNγ was referred to Example 16. The experimental results were as Figure 13 shown. The humanized antibodies 864F3-Hu4, 864F3-Hu5, 871G1-Hu1, 871G1-Hu2, 874F7-Hu1, and 874F7-Hu2 had inhibitory effects on IL-33-induced IFNγ secretion by NK cells, and the IC 50 values were 0.904 nM, 1.021 nM, 0.851 nM, 0.900 nM, 0.742 nM, and 1.559 nM, respectively.

[0335] Example 18 Inhibitory effects of humanized antibodies on IL-33-induced IL-5 and IL-13 secretion by KU812 cells

[0336] Basophilic leukemia cells KU812 in the logarithmic growth phase were collected, centrifuged, counted, resuspended with IMDM medium containing 20% FBS (complete medium, purchased from Gibco, catalog number 11965-092), and 96-well cell culture plates were seeded with 20,000 cells per well. Each antibody to be tested was prepared with complete medium and diluted in a 3-fold gradient as the dosing group, and each group was serially diluted for 9 gradients. The highest working concentration of the antibody was 200 μg / mL. At the same time, IL-33-his was prepared with complete medium at a working concentration of 700 ng / ml. After mixing at a ratio of 1:1, it was added to the above 96-well cell culture plates, and the final volume per well was 200 μL. The non-dosing group was set as the negative control, and the group with only IL-33 added was set as the single-drug control group. Two replicates were made for each concentration, and the cells were further cultured in a 37°C cell culture incubator for 48 h. The supernatant was collected to measure the secretion amounts of IL-5 and IL-13.

[0337] Anti-human IL-5 (purchased from BD, catalog number 554488) and anti-human IL-13 (purchased from BD, catalog number 554570) antibodies were diluted to 5ug / ml with ELISA coating solution, coated on ELISA plates, 100μL / well, placed in a humidified box, 4°C, and coated for 16h. Wash the ELISA plate three times with PBST to remove unbound antigens, and pat the ELISA plate dry on absorbent paper to remove excess liquid, then block with 2% BSA prepared in PBS, 200μL / well, at room temperature for 1h. Wash once with PBST to remove excess blocking solution, pat the ELISA plate dry, remove excess liquid, and dilute the hu-IL-5 standard with 1% BSA prepared in PBST in a 3-fold gradient, with the highest concentration of 50ng / mL, dilute 12 gradients, add to ELISA wells, 100μL / well, incubate at room temperature for 1h, and make 2 replicates for each sample in parallel. Add 100 μL / well of the collected cell supernatant. Dilute the hu-IL-13 standard in 3-fold gradients with 1% BSA prepared in PBST, with the highest concentration being 10 ng / mL. Dilute 12 gradients and add to the ELISA wells at 100 μL / well. Incubate at room temperature for 2 hours. Make 2 replicates for each sample in parallel. Add 50 μL / well of the collected cell supernatant. Wash away the unbound or non-specifically bound primary antibody. According to the antibody instructions, dilute the Biotin-anti-human-IL-5 (purchased from BD, catalog number 554491) and Biotin-anti-human-IL-13 (purchased from BD, catalog number 555054) secondary antibodies to the appropriate concentrations with antibody diluent, add to the ELISA plate at 100 μL / well, and incubate at room temperature for 2 hours. Wash three times with PBST, pat the ELISA plate dry on absorbent paper, remove excess liquid, dilute HRP-labeled Streptavidin (purchased from BD, catalog number 554066) to an appropriate concentration with antibody diluent, add ELISA plate, 100 μL / well, and incubate at room temperature for 0.5 h. Wash three times with PBST, pat the ELISA plate dry on absorbent paper, remove excess liquid, add TMB colorimetric solution, 100 μL / well, color to an appropriate depth, add 2M H2SO4, 50 μL / well to stop color development, and measure its absorbance at a wavelength of 450 nm in a multifunctional microplate reader to analyze the data.

[0338] The experimental results are as follows Figure 14 As shown in Figure 2, humanized antibodies 864F3-Hu4 and 874F7-Hu1 could effectively inhibit IL-33-induced IL-5 secretion from KU812 cells, with IC 50 27.605nM and 7.828nM respectively.

[0339] likeFigure 15 As shown, the humanized antibodies 864F3-Hu4 and 874F7-Hu1 can also effectively inhibit the secretion of IL-13 by KU812 cells induced by IL-33, and their IC 50 values are 14.745 nM and 5.219 nM, respectively.

[0340] Example 19 Cross-reactivity of Humanized Antibodies to IL-33 Proteins of Different Species

[0341] The experimental method was referred to Example 14, in which the antigens were cynomolgus monkey IL-33 protein (purchased from Sinobiological, catalog number 90912-CNAE) and mouse IL-33 protein (purchased from R&D, 3626-ML-010 / CF), and human IL-33 protein was set as a control at the same time. The experimental results are shown in Table 3. Both 864F3-Hu4 and 874F7-Hu1 have cross-reactivity with cynomolgus monkey IL-33 protein, and are consistent with the binding characteristics of each antibody to human IL-33 protein. In addition, 864F3-Hu4 and 874F7-Hu1 also have certain cross-reactivity with mouse IL-33 protein, but their affinity is significantly lower than the binding of each antibody to human IL-33 protein.

[0342] Table 3: Cross-reactivity of Humanized Antibodies to Cynomolgus Monkey and Mouse IL-33 Proteins

[0343]

[0344] Example 20 Evaluation of In Vivo Neutralizing Activity of Humanized Antibodies

[0345] The specific experimental procedure was referred to Example 7, with 10 BALB / C mice in each experimental group.

[0346] As Figure 16 shown, the results indicate that compared with the control group (spleen weight 80.55 mg), the spleen in the IL-33 antigen group was significantly heavier, at 176.49 mg. The average spleen weights of the antibody administration groups of 864F3-Hu4, 864F3-Hu5, 871G1-Hu1, 871G1-Hu2, 874F7-Hu1, and 874F7-Hu2 were 78.39 mg, 86.6 mg, 92.82 mg, 98.87 mg, 75.63 mg, and 77.49 mg, respectively. It can be seen that each humanized antibody has obvious neutralizing activity in vivo.

[0347] Example 21 Determination of In Vivo Pharmacodynamic Activity of Humanized Antibodies

[0348] The experimental method was referred to Example 7, and the experimental results are as Figure 17As shown, after intraperitoneal injection of human IL-33, it can significantly stimulate splenomegaly in mice (IL-33 group), with the spleen weight being approximately 194.6 mg, while the control group (without human IL-33 stimulation) was 76.1 mg. When 5 mg / kg of 864F3-Hu4 and 874F7-Hu1 were given for single treatment respectively, it could effectively inhibit splenomegaly in mice, with the spleen weights being 65.4 mg and 55.9 mg respectively.

[0349] At the end of the above experiment, the mice in each group were euthanized, and the levels of eosinophils and IL-5 in peripheral blood were detected respectively.

[0350] A part of the whole blood of the above mice was taken out and placed in a common EP tube, and left at 4°C for more than 5 h 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, the serum was taken out and placed in a new centrifuge tube, and then centrifuged again at 8000 rmp for 6 min in a centrifuge pre-cooled at 4°C. After centrifugation, the supernatant was taken out, aliquoted, and stored at -80. The standards and the serum samples to be tested were diluted according to the instructions of BD CBA Mouse Enhanced Sensivity Master Buffer Kit (purchased from BD biosciences, catalog number 562246). Subsequently, each sample was processed according to the instructions of Mouse IL-5 Enhanced Sensitivity Flex Set (purchased from BD biosciences, catalog number 562234). The detection parameters were set, and each sample was detected on a flow cytometer (BD FACSCelesta). The data collected above were analyzed by GraphPad Prism, and the content of IL-5 in the serum samples of each experimental group was calculated according to the standard curve.

[0351] The experimental results are as Figure 18 shown. Compared with the control group (without human IL-33 stimulation), after intraperitoneal injection of human IL-33, it can significantly stimulate the secretion of IL-5 in the peripheral blood of mice (IL-33 group), about 78.6 times. When 5 mg / kg of 864F3-Hu4 and 874F7-Hu1 were given for single treatment respectively, it could effectively inhibit the secretion of IL-5 in the peripheral blood of mice, which were 1.7 times and 0.9 times that of the control group respectively.

[0352] Take a part of the above-mentioned mouse whole blood and place it in an EDTA anticoagulation tube. Invert it up and down evenly and store it at 4°C for later use. Take a 10 mL round-bottom centrifuge tube and add 2 μL of mouse Fc blocker (purchased from BD biosciences, catalog number 553142) to each tube. Take 50 μL of blood sample from each sample and place it in a 10 mL centrifuge tube. Gently pipette to mix the blood sample and the blocker evenly. Incubate on ice for 15 min. Add 2 μL of the mixed three antibodies to each centrifuge tube: 0.5 μL of CD45.2 MonoclonalAntibody(104), PerCP-Cyanine5.5 (purchased from eBioscience TM , catalog number 45-0454-82), 0.5 μL of CD170 (Siglec F) Monoclonal Antibody(1RNM44N), PE (purchased from eBioscience TM , catalog number 12-1702-82), 1 μL of Ly-6G / Ly - 6C Monoclonal Antibody(RB6-8C5), APC-eFluor 780 (purchased from eBioscience TM , catalog number 47-5931-82); Incubate in the dark on ice for 1 h. Add 500 mL of 1x Lysing buffer (purchased from BD Biosciences, catalog number 555899) to the centrifuge tube, mix well, and lyse red blood cells at room temperature for 5 min. Centrifuge the centrifuge tube at 350 g for 5 min. Discard the supernatant, add 3 mL of pre-cooled PBS to the tube to wash the cells, and centrifuge at 350 g for 5 min. Discard the supernatant, add 500 μL of pre-cooled and filtered PBS to the tube, and gently flick to resuspend the sedimented cells. Filter the cell suspension through a cell sieve into a flow cytometry tube, and analyze the proportion of eosinophils in white blood cells in each sample by flow cytometry.

[0353] The experimental results are as Figure 19 shown. Compared with the control group (without human IL-33 stimulation), after intraperitoneal injection of human IL-33, it can significantly stimulate the increase of eosinophils in the peripheral blood of mice (IL-33 group), about 10.5 times. When 5 mg / kg of 864F3-Hu4 and 874F7-Hu1 are given for single treatment respectively, it can effectively reduce the increase of eosinophils in the peripheral blood of mice caused by human IL-33 stimulation, which are 1.6 times and 1.58 times that of the control group respectively.

[0354] Example 22 Determination of the binding epitope of the humanized antibody to IL-33

[0355] According to the amino acid sequence of the mature protein of IL-33:

[0356] MSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET (SEQ ID NO.55) (The amino acid sequence is from Ser at position 112 to Thr at position 270 of NCBI: NP_254274.1). Sixteen polypeptides were synthesized to characterize the binding epitopes of the humanized antibody to IL-33. The amino acid sequences of each polypeptide are shown in Table 4, and the N-terminus of each was biotinylated. Table 4: Amino acid sequences of polypeptides

[0357]

[0358]

[0359] To determine the binding activity of the antibody to each polypeptide fragment, biotin-avidin (SA) was pre-diluted to 2 μg / mL with coating buffer (50 mM carbonate coating buffer, pH 9.6), 100 μL per well, and used to coat ELISA plates at 4 °C overnight; washed 3 times with PBST, then blocked with 2% BSA prepared with PBS at room temperature for 2 h, 200 μL per well. Washed once with PBST, and 864F3 and 874F7 with a final concentration of 10 μg / mL were added respectively, 100 μL per well, and incubated at room temperature for 1 h. Washed 3 times with PBST, added HRP-labeled goat anti-mouse IgG secondary antibody, and incubated at room temperature for 30 min; after washing the plates 3 times with PBST, the residual liquid drops were patted dry as much as possible on absorbent paper, 100 μL of TMB chromogenic solution was added to each well, and the color development was allowed to reach an appropriate depth; 50 μL of 2 M H2SO4 termination solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader to analyze the binding ability of the antibody to be tested to each polypeptide fragment. The experimental results are shown in Table 5. It can be seen that 874F7 can significantly bind to Pep6 and not to other peptide segments.

[0360] Table 5: Amino acid sequences of polypeptides

[0361]

[0362]

[0363] Based on the above experimental results, and the binding Kd value of 874F7-Hu1 to murine IL-33 protein is 3.32×10 -8; according to the amino acid sequence of the above-mentioned mature IL-33 protein, select:

[0364] The region of "KKDEKKDKVLLSYYESQHPSNESGDGVDGK" (positions 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 IL-33 mutant proteins with amino acid site mutations as follows:

[0365] 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.

[0366] Subsequently, referring to the experimental method of Example 9, the affinities of the above mutant proteins for 874F7-Hu1 were measured respectively, and IL-33 proteins without any mutations (WT-1, WT-2, WT-3, WT-4) were set as controls.

[0367] Representative experimental results are respectively as Figures 20 - 23 and Table 6 show that compared with WT, after mutations at K45, V49, L50, and D65, the affinity of 874F7-Hu1 for IL-33 was significantly weakened, decreasing by more than 25-fold; after mutations at S60 and S52, the affinity of 874F7-Hu1 for IL-33 was significantly weakened, decreasing by 10 - 25-fold; after mutations at K48, L51, Y53, and E55, the affinity of 874F7-Hu1 for IL-33 was somewhat weakened, decreasing by 2.5 - 10-fold. This also indicates that the key sites affecting the binding of 874F7-Hu1 to IL-33 are mainly K45, V49, D65, and L50, followed by S60 and S52, and then K48, L51, Y53, and E55.

[0368] The positions of the above sites in the IL-33 crystal 3D structure diagram (derived from PDB: 2KLL) are as Figure 24 shown, which further indicates that the binding epitope of 874F7-Hu1 to IL-33 is a linear and spatial epitope including the above key amino acid sites.

[0369] Table 6: Binding affinities of 874F7-Hu1 to various mutant proteins of IL-33

[0370]

[0371]

[0372] All documents mentioned in the present invention are incorporated herein by reference as if each individual document was specifically and individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application. Sequence Listing <110> 3SBio Inc. <120> Antibody Binding to Human IL-33, Its Preparation Method and Use <130> P2021-2351 <150> 2020110233018 <151> 2020-09-25 <160> 71 <170> PatentIn version 3.5 <210> 1 <211> 363 <212> DNA <213> Mus musculus <400> 1 caggtacagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc 60 acttgcactg tctctgggtt ttcattaacc aactatggtg tacactgggt tcgcctgcct 120 ccaggaaagg gtctggagtg gctgggagtg atacgagctg gtggaagttc agattataat 180 tcggctctca tgtccagact gaacatcagg aaagacaatt ccaagagcca agttttctta 240 gaaatgaaca gtcttcaaac tgctgacaca gccatgtact actgtgccag agaccattat 300 ttcagtaata gttacggggg ttctccttac tggggccaag ggactctggt cactgtctct 360 gca 363 <210> 2 <211> 121 <212> PRT <213> Mus musculus <400> 2 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Leu Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Arg Ala Gly Gly Ser Ser Asp Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Asn Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Asn Ser Leu Gln Thr Ala Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Asp His Tyr Phe Ser Asn Ser Tyr Gly Gly Ser Pro Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 3 <211> 363 <212> DNA <213> Mus musculus <400> 3 caggttcacc tgaaggagtc agggcctggc ctggtggcgc cctcacagag cctgtccatc 60 Gln Val His Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln acttgcaatg tctctggatt ttcattatcc aaatatggtg tacactggat ccgccagact 120 Thr Cys Asn Val Ser Gly Phe Ser Leu Ser Lys Tyr Gly Val His Trp Ile ccaggaaggg gtctggactg gctgggagtg ttacgggctg gtggaaccat aagttataat 180 Arg Gln Thr Pro Gly Arg Gly Leu Asp Trp Leu Gly Val Leu Arg Ala Gly tcggctctca tgtccagact gagtatcagc gaagacattt ccaaaagcca agttttctta 240 Ser Tyr Asn Ser Ala Leu Met Ser Leu Ser Ile Thr Cys Asn Val Ser Gly aaaatgaatg atttacaaac tgatgactca gccatctact tctgtgccag agaccattac 300 Lys Glu Met Ile Tyr Asn Leu Asp Asp Ser Ala Ile Thr Leu Cys Gln Arg tactatagtt ccttcggggg ttttgcttcc tggggtcagg ggactctggt cactgtctct 360 Tyr Tyr Ser Phe Arg Gly Phe Ala Phe Trp Gly Gln Gly Thr Leu Val Thr gca 363 Ala <210> 4 <211> 121 <212> PRT <213> Mus musculus <400> 4 Gln Val His Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Asn Val Ser Gly Phe Ser Leu Ser Lys Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Thr Pro Gly Arg Gly Leu Asp Trp Leu 35 40 45 Gly Val Leu Arg Ala Gly Gly Thr Ile Ser Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Ser Ile Ser Glu Asp Ile Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Asp Leu Gln Thr Asp Asp Ser Ala Ile Tyr Phe Cys Ala 85 90 95 Arg Asp His Tyr Tyr Tyr Ser Ser Phe Gly Gly Phe Ala Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 5 <211> 321 <212> DNA <213> Mus musculus <400> 5 gacattcaga tgacccagtc tcctgcctcc cagtctgcat ctctgggaga aagtgtcacc 60 atcacatgcc tggcaagtca gaccattgct acatggttag catggtatca gcagaaacca 120 gggaaatctc ctcagctcct gatttatgct gcaaccaggt tggcagatgg ggtcccatca 180 aggttcagtg gtagtggatc tggcacagaa ttttctttca agatcagtag cctacaggct 240 gaagattttg tgatttatta ctgtcaacaa ctttacaata ctccgtacac gttcggaggg 300 gggaccaagc tggagataaa a 321 <210> 6 <211> 107 <212> PRT <213> Mus musculus <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly 1 5 10 15 Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Ala Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ala Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Ser Phe Lys Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Phe Val Ile Tyr Tyr Cys Gln Gln Leu Tyr Asn Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 363 <212> DNA <213> Mus musculus <400> 7 caggtgcaac tgaaggagtc aggacctggt ctggtggcgc cctcacacag cctgtccatc 60 acctgcaatg tctctgggtt ttcattatcc aagtatggtg tacactggat tcgtcagatt 120 ccaggaaggg gtctggactg gctgggagtg ttacgggctg gtggaagtac aggttataat 180 tcggctctca tgtccaggct gagtatcagc aaagacagtt ccaaaagcca agttttctta 240 aagatgaacg atctacggac tgatgacaca gccgtttact tctgtgtcag agaccattac 300 tactacagtt cttacggggg ttttgtttac tggggccagg ggactctggt cactgtctct 360 gca 363 <210> 8 <211> 121 <212> PRT <213> Mus musculus <400> 8 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser His 1 5 10 15 Ser Leu Ser Ile Thr Cys Asn Val Ser Gly Phe Ser Leu Ser Lys Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Ile Pro Gly Arg Gly Leu Asp Trp Leu 35 40 45 Gly Val Leu Arg Ala Gly Gly Ser Thr Gly Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Asp Leu Arg Thr Asp Asp Thr Ala Val Tyr Phe Cys Val 85 90 95 Arg Asp His Tyr Tyr Tyr Ser Ser Tyr Gly Gly Phe Val Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 9 <211> 318 <212> DNA <213> Mus musculus <400> 9 gacattcaga tgacccagtc tcctgcctcc cagtctgcat ctctgggaga aagtgtcacc 60 atcacatgcc tggcaagtca gaccataggt gcatggttag catggtatcg gcagcaacca 120 ggaaaatctc ctcagctcct gatttatgct gcaaccaggt tggcagatgg ggtcccatca 180 aggttcagtg gtagtggttc tgggacagaa ttttctttca agatcaacaa cctacaggct 240 gaagattttg taagttatta ctgtcaacaa cttgacagta gtccgtacac gttcggaggg 300 gggaccaggc tggaaatg 318 <210> 10 <211> 106 <212> PRT <213> Mus musculus <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly 1 5 10 15 Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Ala Trp 20 25 30 Leu Ala Trp Tyr Arg Gln Gln Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ala Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Ser Phe Lys Ile Asn Asn Leu Gln Ala 65 70 75 80 Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Asp Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Met 100 105 <210> 11 <211> 318 <212> DNA <213> Mus musculus <400> 11 gacattcaga tgacccagtc tcctgcctcc cagtctgcat ctctgggaga aagtgtcacc 60 atcacatgcc tggcaagtca gaccattggt gcatggttag catggtatcg gcagcaacca 120 ggaaaatctc ctcagctcct gatttatgct gcaaccaggt tggcagatgg ggtcccatca 180 aggttcagtg gtagtggatc tggcacaaaa ttttctttca agatcaacaa cctacaggct 240 gaagattttg tgatttatta ctgtcaacaa cttaacagta ctccgtacac gttcggaggg 300 gggaccaggc tggaaatg 318 <210> 12 <211> 106 <212> PRT <213> Mus musculus <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly 1 5 10 15 Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Ala Trp 20 25 30 Leu Ala Trp Tyr Arg Gln Gln Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ala Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Asn Asn Leu Gln Ala 65 70 75 80 Glu Asp Phe Val Ile Tyr Tyr Cys Gln Gln Leu Asn Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Met 100 105 <210> 13 <211> 327 <212> PRT <213> Human <400> 13 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 14 <211> 107 <212> PRT <213> Human <400> 14 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 15 <211> 5 <212> PRT <213> Mus musculus <400> 15 Asn Tyr Gly Val His 1 5 <210> 16 <211> 16 <212> PRT <213> Mus musculus <400> 16 Val Ile Arg Ala Gly Gly Ser Ser Asp Tyr Asn Ser Ala Leu Met Ser 1 5 10 15 <210> 17 <211> 13 <212> PRT <213> Mus musculus <400> 17 Asp His Tyr Phe Ser Asn Ser Tyr Gly Gly Ser Pro Tyr 1 5 10 <210> 18 <211> 5 <212> PRT <213> Mus musculus <400> 18 Lys Tyr Gly Val His 1 5 <210> 19 <211> 16 <212> PRT <213> Mus musculus <400> 19 Val Leu Arg Ala Gly Gly Thr Ile Ser Tyr Asn Ser Ala Leu Met Ser 1 5 10 15 <210> 20 <211> 13 <212> PRT <213> Mus musculus <400> 20 Asp His Tyr Tyr Tyr Ser Ser Phe Gly Gly Phe Ala Ser 1 5 10 <210> 21 <211> 11 <212> PRT <213> Mus musculus <400> 21 Leu Ala Ser Gln Thr Ile Ala Thr Trp Leu Ala 1 5 10 <210> 22 <211> 7 <212> PRT <213> Mus musculus <400> 22 Ala Ala Thr Arg Leu Ala Asp 1 5 <210> 23 <211> 9 <212> PRT <213> Mus musculus <400> 23 Gln Gln Leu Tyr Asn Thr Pro Tyr Thr 1 5 <210> 24 <211> 16 <212> PRT <213> Mus musculus <400> 24 Val Leu Arg Ala Gly Gly Ser Thr Gly Tyr Asn Ser Ala Leu Met Ser 1 5 10 15 <210> 25 <211> 13 <212> PRT <213> Mus musculus <400> 25 Asp His Tyr Tyr Tyr Ser Ser Tyr Gly Gly Phe Val Tyr 1 5 10 <210> 26 <211> 11 <212> PRT <213> Mus musculus <400> 26 Leu Ala Ser Gln Thr Ile Gly Ala Trp Leu Ala 1 5 10 <210> 27 <211> 9 <212> PRT <213> Mus musculus <400> 27 Gln Gln Leu Asp Ser Ser Pro Tyr Thr 1 5 <210> 28 <211> 9 <212> PRT <213> Mus musculus <400> 28 Gln Gln Leu Asn Ser Thr Pro Tyr Thr 1 5 <210> 29 <211> 121 <212> PRT <213> Composite <400> 29 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Arg Ala Gly Gly Ser Ser Asp Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp His Tyr Phe Ser Asn Ser Tyr Gly Gly Ser Pro Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 30 <211> 121 <212> PRT <213> Composite <400> 30 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Ile Arg Leu Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Arg Ala Gly Gly Ser Ser Asp Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp His Tyr Phe Ser Asn Ser Tyr Gly Gly Ser Pro Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 31 <211> 121 <212> PRT <213> Composite <400> 31 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Leu Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Arg Ala Gly Gly Ser Ser Asp Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp His Tyr Phe Ser Asn Ser Tyr Gly Gly Ser Pro Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 32 <211> 107 <212> PRT <213> Composite <400> 32 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Ala Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Tyr Asn Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 33 <211> 121 <212> PRT <213> Composite <400> 33 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Lys Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Leu Arg Ala Gly Gly Thr Ile Ser Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp His Tyr Tyr Tyr Ser Ser Phe Gly Gly Phe Ala Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 34 <211> 121 <212> PRT <213> Composite <400> 34 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Lys Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Leu Arg Ala Gly Gly Thr Ile Ser Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Val Thr Ile Ser Glu Asp Ile Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp His Tyr Tyr Tyr Ser Ser Phe Gly Gly Phe Ala Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 35 <211> 121 <212> PRT <213> Composite <400> 35 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Lys Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Leu Arg Ala Gly Gly Thr Ile Ser Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Thr Ile Ser Glu Asp Ile Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp His Tyr Tyr Tyr Ser Ser Phe Gly Gly Phe Ala Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 36 <211> 107 <212> PRT <213> Composite <400> 36 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Ala Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 37 <211> 121 <212> PRT <213> Composite <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Lys Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Leu Arg Ala Gly Gly Ser Thr Gly Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp His Tyr Tyr Tyr Ser Ser Tyr Gly Gly Phe Val Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 121 <212> PRT <213> Composite <400> 38 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Lys Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Leu Arg Ala Gly Gly Ser Thr Gly Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp His Tyr Tyr Tyr Ser Ser Tyr Gly Gly Phe Val Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 39 <211> 121 <212> PRT <213> Composite <400> 39 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Lys Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Leu Arg Ala Gly Gly Ser Thr Gly Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Arg Asp His Tyr Tyr Tyr Ser Ser Tyr Gly Gly Phe Val Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 40 <211> 107 <212> PRT <213> Composite <400> 40 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Ala Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asp Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 41 <211> 363 <212> DNA <213> Composite <400> 41 caggtgcagc tgcaggagtc tggaccaggc ctggtgaagc cttctgagac cctgagcctg 60 acctgtaccg tgtccggctt tagcctgaca aattatggcg tgcattggat tagacagcca 120 cctggcaagg gcctggagtg gattggcgtg attagagctg gaggttcctc tgattataat 180 tctgctctga tgtctagggt gaccattagt gtggatacat ctaagaatca gttttctctg 240 aagctgagtt ctgtgactgc cgctgataca gctgtgtatt attgtgctag ggaccattat 300 ttttctaata gctacggcgg cagtccttac tggggccagg gcacactggt gaccgtgtct 360 agc 363 <210> 42 <211> 363 <212> DNA <213> Composite <400> 42 caggtgcagc tgcaggagag cggccctgga ctggtgaagc catctgagac tctgagcctg 60 acctgtacag tgagcggatt ttctctgact aattacggag tgcattggat tagactgcct 120 ccaggaaagg gcctggaatg gatcggtgtg attagagccg gaggctcctc tgattataat 180 tccgctttga tgtctagagt gaccatctct aaagataact ctaagagtca ggtgtctctg 240 aagctgagta gcgtgacagc agctgatacc gctgtgtatt actgcgccag ggatcattac 300 ttttcaaact cttatggagg atctccttac tggggccagg gcaccctggt gacagtgagc 360 tct 363 <210> 43 <211> 363 <212> DNA <213> Composite <400> 43 caggtgcagc tgcaggagtc tggccccggc ctggtgaagc cctccgagac cctgagcctg 60 acttgcactg tgagtggctt tagtctgact aattatggcg tgcattgggt gaggctgcct 120 ccaggcaagg gactggagtg gctgggagtg attagagctg gtggatctag tgattataac 180 tccgctctga tgtccagact gaccatctct aaggacaact ccaagagcca ggtgtccctg 240 aagctgtcta gtgtgaccgc cgccgacacc gccgtgtact actgcgccag ggaccactac 300 ttttctaact cttacggggg ctccccctac tggggccagg gcaccttggt gacagtgtct 360 agc 363 <210> 44 <211> 321 <212> DNA <213> Composite <400> 44 gatattcaga tgacacagtc tccttcttct ctgtctgctt ctgtgggaga tagagtgacc 60 attacctgcc tggcttctca gaccattgcc acatggctgg cttggtatca gcagaagcct 120 ggaaagagcc ctaagctgct gatctatgct gctacaagac tggctgatgg cgtgccatct 180 agattctccg gatctggatc cggaacagac ttcaccctga caatctcttc tctgcagcct 240 gaggatttcg ctacctacta ttgccagcag ctgtataata ctccttatac atttggccag 300 ggcaccaagg tcgaaatcaa g 321 <210> 45 <211> 363 <212> DNA <213> Composite <400> 45 caggtgcagc tgcaggaatc tggacctgga ctggtgaagc catccgagac actgtctctg 60 acatgtacag tgtctggctt ttctctgtct aagtatggag tgcattggat cagacagcct 120 cctggcaagg gcctggagtg gattggagtg ctgagagctg gcggaacaat tagttataat 180 tctgctctga tgtctagagt gaccatcagc gtggatacct ctaagaacca gttttctctg 240 aagctgagtt ctgtgacagc agccgacact gctgtgtact actgtgctcg cgaccactac 300 tactacagct ctttcggcgg cttcgcctcc tgggggcagg gcaccctggt gaccgtgtcc 360 tcc 363 <210> 46 <211> 363 <212> DNA <213> Composite <400> 46 caggtgcagc tgcaggagtc cggccctggc ctggtgaagc cctccgagac cctgagcctg 60 acctgcaccg tgtccggctt ctccctgtcc aagtacggcg tgcactggat caggcagcct 120 cccggcaagg gcctggagtg gattggcgtt ctgagagctg gaggcactat ttcttataat 180 agcgcactga tgtctagagt gacaatcagt gaggatatct ctaagtccca ggtgagcctg 240 aagttaagtt ctgtgacagc tgctgacacc gctgtgtatt attgtgctag ggatcattac 300 tactactctt ccttcggcgg ctttgcctct tggggccagg gcaccctggt taccgtgagt 360 agt 363 <210> 47 <211> 363 <212> DNA <213> Composite <400> 47 caggtgcagc tgcaggagag cggccccggc ctggtgaagc cctccgagac cctgagcctg 60 acctgcaccg tgtccggctt ttccctgagc aagtacggcg tgcactggat ccgccagccc 120 cccggcaagg gcctggagtg gctgggcgtg ctgagggccg gcggcaccat cagctacaac 180 tccgccctga tgtcccggct gaccatctct gaggacatct ccaagtccca ggtgtccctg 240 aagctgagct ccgtgaccgc cgccgacacc gccgtgtact actgcgcccg cgaccactac 300 tactacagca gcttcggcgg ctttgccagc tggggacagg gcaccctggt gaccgtgagc 360 agc 363 <210> 48 <211> 321 <212> DNA <213> Composite <400> 48 gacatccaga tgacccagtc cccctcctcc gtgtcggcca gcgtgggcga cagagtgacc 60 atcacctgcc tggcctccca gacgatcggc gcctggctgg cctggtacca gcagaagccc 120 ggcaagtccc ccaagctgct gatctacgcc gccaccagac tggccgacgg cgtgccctcc 180 aggttcagcg gctccggatc tggtaccgat tttaccctga caatctctag cctgcagcct 240 gaggattttg ccacatacta ctgtcagcag ctgaatagta ctccatacac ctttggccag 300 ggaaccaagg tggagatcaa g 321 <210> 49 <211> 363 <212> DNA <213> Composite <400> 49 caggtgcagc tgcaggagtc cggccccggc ctggtgaagc catccgagac cctgagcctt 60 acctgcaccg tgtctggctt cagcctgtct aagtacggcg tgcattggat caggcagccc 120 cccggcaagg gcctggagtg gatcggcgtg ctgagagccg gcggcagcac aggctacaac 180 tccgccctga tgagcagggt gaccatcagc gtggacacca gcaagaacca gtttagcctg 240 aagctgagca gcgtgaccgc cgccgacacc gccgtgtact actgcgctcg ggaccactac 300 tactactcca gctacggcgg cttcgtgtac tggggccagg gcaccctggt gaccgtgtct 360 agc 363 <210> 50 <211> 363 <212> DNA <213> Composite <400> 50 caggtgcagc tgcaggagtc tggccctgga ctggtgaagc cttctgagac actgagtctg 60 acatgtacag tgagtggctt ctctctgtcg aagtatggag tgcactggat taggcagccc 120 cctggaaagg gcctggagtg gatcggcgtg ctgcgggccg gcggctctac gggctacaac 180 tccgccctga tgagccgggt gaccatcagc aaggacacct ccaagagcca ggtgagcctg 240 aagctgtcta gcgtgaccgc cgccgacacc gccgtgtact actgcgccag ggaccactac 300 tactattcca gctatggcgg cttcgtgtac tggggccagg gcaccctggt gaccgtgtcc 360 tcc 363 <210> 51 <211> 363 <212> DNA <213> Composite <400> 51 caggtgcagc tgcaggagag cggcccaggc ctggtgaagc catctgaaac cctgtctctg 60 acctgtacag tgtccggctt ttccctgtct aaatacggtg tgcactggat cagacagcct 120 cctggtaaag gcctggagtg gctgggagtg ctgagggctg gaggaagtac cggctataat 180 agcgctctga tgtctagatt gactatctct aaggatagct ctaaatctca ggtgagcctg 240 aaactgagct ctgtcaccgc tgccgatacc gctgtttatt attgtgtgag ggatcattac 300 tactactcct cttacggagg atttgtgtat tggggtcagg gcacactggt gacagtctcc 360 tct 363 <210> 52 <211> 321 <212> DNA <213> Composite <400> 52 gacatccaga tgacacagtc tcctagctct gtgagcgctt ctgtgggaga tagagtgaca 60 atcacctgtc tggcttctca gaccattgga gcttggctgg cttggtacca gcagaaacca 120 ggcaagtctc ctaagctgct gatctatgct gctactagac tggctgatgg cgtgccatct 180 agattttctg gcagcggatc tggaaccgat tttacactga caatctcttc tctgcagcct 240 gaggatttcg ctacatacta ttgtcagcag ctggattctt ctccttacac atttggccag 300 ggaacaaagg tggaaattaa g 321 <210> 53 <211> 166 <212> PRT <213> Composite <400> 53 Met Ser Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu 1 5 10 15 Ser Thr Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser 20 25 30 Tyr Glu Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp Lys 35 40 45 Val Leu Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly 50 55 60 Asp Gly Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys 65 70 75 80 Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His 85 90 95 Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn 100 105 110 Met His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val 115 120 125 Phe Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser 130 135 140 Ser Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr 145 150 155 160 His His His His His His 165 <210> 54 <211> 560 <212> PRT <213> Composite <400> 54 Met Gly Phe Trp Ile Leu Ala Ile Leu Thr Ile Leu Met Tyr Ser Thr 1 5 10 15 Ala Ala Lys Phe Ser Lys Gln Ser Trp Gly Leu Glu Asn Glu Ala Leu 20 25 30 Ile Val Arg Cys Pro Arg Gln Gly Lys Pro Ser Tyr Thr Val Asp Trp 35 40 45 Tyr Tyr Ser Gln Thr Asn Lys Ser Ile Pro Thr Gln Glu Arg Asn Arg 50 55 60 Val Phe Ala Ser Gly Gln Leu Leu Lys Phe Leu Pro Ala Ala Val Ala 65 70 75 80 Asp Ser Gly Ile Tyr Thr Cys Ile Val Arg Ser Pro Thr Phe Asn Arg 85 90 95 Thr Gly Tyr Ala Asn Val Thr Ile Tyr Lys Lys Gln Ser Asp Cys Asn 100 105 110 Val Pro Asp Tyr Leu Met Tyr Ser Thr Val Ser Gly Ser Glu Lys Asn 115 120 125 Ser Lys Ile Tyr Cys Pro Thr Ile Asp Leu Tyr Asn Trp Thr Ala Pro 130 135 140 Leu Glu Trp Phe Lys Asn Cys Gln Ala Leu Gln Gly Ser Arg Tyr Arg 145 150 155 160 Ala His Lys Ser Phe Leu Val Ile Asp Asn Val Met Thr Glu Asp Ala 165 170 175 Gly Asp Tyr Thr Cys Lys Phe Ile His Asn Glu Asn Gly Ala Asn Tyr 180 185 190 Ser Val Thr Ala Thr Arg Ser Phe Thr Val Lys Asp Glu Gln Gly Phe 195 200 205 Ser Leu Phe Pro Val Ile Gly Ala Pro Ala Gln Asn Glu Ile Lys Glu 210 215 220 Val Glu Ile Gly Lys Asn Ala Asn Leu Thr Cys Ser Ala Cys Phe Gly 225 230 235 240 Lys Gly Thr Gln Phe Leu Ala Ala Val Leu Trp Gln Leu Asn Gly Thr 245 250 255 Lys Ile Thr Asp Phe Gly Glu Pro Arg Ile Gln Gln Glu Glu Gly Gln 260 265 270 Asn Gln Ser Phe Ser Asn Gly Leu Ala Cys Leu Asp Met Val Leu Arg 275 280 285 Ile Ala Asp Val Lys Glu Glu Asp Leu Leu Leu Gln Tyr Asp Cys Leu 290 295 300 Ala Leu Asn Leu His Gly Leu Arg Arg His Thr Val Arg Leu Ser Arg 305 310 315 320 Lys Asn Pro Ser Lys Glu Cys Phe Glu Asn Leu Tyr Phe Gln Gly Thr 325 330 335 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 340 345 350 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 355 360 365 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 370 375 380 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 385 390 395 400 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 405 410 415 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 420 425 430 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 435 440 445 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 450 455 460 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 465 470 475 480 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 485 490 495 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 500 505 510 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 515 520 525 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 530 535 540 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 545 550 555 560 <210> 55 <211> 160 <212> PRT <213> human <400> 55 Met Ser Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu 1 5 10 15 Ser Thr Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser 20 25 30 Tyr Glu Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp Lys 35 40 45 Val Leu Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly 50 55 60 Asp Gly Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys 65 70 75 80 Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His 85 90 95 Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn 100 105 110 Met His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val 115 120 125 Phe Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser 130 135 140 Ser Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr 145 150 155 160 <210> 56 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep1 <400> 56 Val Gln Lys Tyr Thr Arg Ala Leu His Asp Ser Ser Ile Thr Gly Ile 1 5 10 15 Ser Pro Ile Thr 20 <210> 57 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep2 <400> 57 Ser Ser Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu 1 5 10 15 Ser Thr Tyr Asn 20 <210> 58 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep3 <400> 58 Glu Tyr Leu Ala Ser Leu Ser Thr Tyr Asn Asp Gln Ser Ile Thr Phe 1 5 10 15 Ala Leu Glu Asp 20 <210> 59 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep4 <400> 59 Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser Tyr Glu Ile Tyr 1 5 10 15 Val Glu Asp Leu 20 <210> 60 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep5 <400> 60 Glu Ser Tyr Glu Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys 1 5 10 15 Asp Lys Val Leu 20 <210> 61 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep6 <400> 61 Lys Lys Asp Glu Lys Lys Asp Lys Val Leu Leu Ser Tyr Tyr Glu Ser 1 5 10 15 Gln His Pro Ser 20 <210> 62 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep7 <400> 62 Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly Asp Gly 1 5 10 15 Val Asp Gly Lys 20 <210> 63 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep8 <400> 63 Asn Glu Ser Gly Asp Gly Val Asp Gly Lys Met Leu Met Val Thr Leu 1 5 10 15 Ser Pro Thr Lys 20 <210> 64 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep9 <400> 64 Met Leu Met Val Thr Leu Ser Pro Thr Lys Asp Phe Trp Leu His Ala 1 5 10 15 Asn Asn Lys Glu 20 <210> 65 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep10 <400> 65 Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His 1 5 10 15 Lys Cys Glu Lys 20 <210> 66 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep11 <400> 66 His Ser Val Glu Leu His Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala 1 5 10 15 Phe Phe Val Leu 20 <210> 67 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep12 <400> 67 Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn Met His Ser Asn 1 5 10 15 Cys Val Ser Phe 20 <210> 68 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep13 <400> 68 His Asn Met His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro 1 5 10 15 Gly Val Phe Ile 20 <210> 69 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep14 <400> 69 Glu Cys Lys Thr Asp Pro Gly Val Phe Ile Gly Val Lys Asp Asn His 1 5 10 15 Leu Ala Leu Ile 20 <210> 70 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep15 <400> 70 Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser Ser Glu 1 5 10 15 Asn Leu Cys Thr 20 <210> 71 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pep16 <400> 71 Lys Val Asp Ser Ser Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys 1 5 10 15 Leu Ser Glu Thr 20

Claims

1. An antibody or an antigen-binding fragment thereof that binds to human IL-33, characterized in that, wherein the affinity of the antibody or its antigen-binding fragment for binding to human IL-33, EC 50 is less than 1 nM; The antibody or its antigen-binding fragment includes: (a3) heavy chain complementarity-determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are shown as SEQ ID NO: 18, 24, and 25 respectively, and (b3) light chain complementarity-determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are shown as SEQ ID NO: 26, 22, and 27 respectively; or (a2) heavy chain complementarity-determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are shown as SEQ ID NO: 18, 19, and 20 respectively, and (b2) light chain complementarity-determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are shown as SEQ ID NO: 21, 22, and 23 respectively; or (a4) heavy chain complementarity-determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are shown as SEQ ID NO: 18, 19, and 20 respectively, and (b4) light chain complementarity-determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are shown as SEQ ID NO: 26, 22, and 28 respectively.

2. The antibody or antigen-binding fragment thereof that binds to human IL-33 according to claim 1, wherein The light chain of the antibody has L-CDR2 shown as SEQ ID No: 22 and has the following characteristics: (t1) Block the binding of IL-33 and the receptor ST2; (t2) Inhibit the secretion of IL-6 by IL-33-induced HUVEC cells; (t3) Inhibit the secretion of IFNγ by human PBMC induced by IL-33 protein; (t4) Inhibit the secretion of IFNγ by IL-33-induced NK cells; and (t5) Inhibit the secretion of IL-5 and IL13 by IL-33-induced KU812 cells.

3. The antibody or antigen-binding fragment thereof that binds to human IL-33 according to claim 1 or 2, characterized in that, Includes: (a) heavy chain complementarity-determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are shown as SEQ ID NO: 18, 24, and 25 respectively, and (b) light chain complementarity-determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are shown as SEQ ID NO: 26, 22, and 27 respectively.

4. The antibody or antigen-binding fragment thereof that binds to human IL-33 according to claim 1 or 2, characterized in that, The antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

5. The antibody or antigen-binding fragment thereof that binds to human IL-33 according to claim 1 or 2, wherein The antigen-binding fragment includes Fab fragment, F(ab’)2 fragment, Fv fragment.

6. The antibody or antigen-binding fragment thereof that binds to human IL-33 according to claim 1, wherein, The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment that binds to human IL-33 are shown as 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.

7. The antibody or antigen-binding fragment thereof that binds to human IL-33 according to claim 1, characterized in that, 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 as SEQ ID NO: 32, and the amino acid sequences of the heavy chain variable region are shown as SEQ ID NO: 33, 34, or 35.

8. The antibody or antigen-binding fragment thereof that binds to human IL-33 according to claim 1, wherein, 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 as SEQ ID NO: 40, and the amino acid sequences of the heavy chain variable region are shown as SEQ ID NO: 37, 38, or 39.

9. The antibody or antigen-binding fragment thereof that binds to human IL-33 according to claim 1, wherein 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 as SEQ ID NO: 36, and the amino acid sequences of the heavy chain variable region are shown as SEQ ID NO: 33, 34, or 35.

10. The antibody or antigen-binding fragment thereof that binds to human IL-33 according to claim 1 or 2, characterized in that, The heavy chain constant region of the antibody is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4.

11. The antibody or antigen-binding fragment thereof that binds to human IL-33 according to claim 10, wherein The amino acid sequences of the heavy chain constant region and the light chain constant region of the antibody or its antigen-binding fragment that binds to human IL-33 are shown as SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

12. The antibody or antigen-binding fragment thereof that binds to human IL-33 according to claim 10, characterized in that, The binding epitope of the antibody or its antigen-binding fragment that binds to human IL-33 and the IL-33 protein contains sites selected from the following group corresponding to SEQ ID NO. 55: Lysine at position 45 (K45), Valine at position 49 (V49), Aspartic acid at position 65 (D65), Leucine at position 50 (L50), Serine at position 60 (S60), Serine at position 52 (S52), Lysine at position 48 (K48), Leucine at position 51 (L51), Tyrosine at position 53 (Y53), Glutamic acid at position 55 (E55).

13. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the antibody or its antigen-binding fragment that binds to human IL-33 as described in any one of claims 1-12.

14. The polynucleotide molecule according to claim 13, wherein, The nucleotide sequences of the polynucleotide molecule encoding the heavy chain variable region and the light chain variable region are shown as SEQ ID NO: 3 and SEQ ID NO: 5, respectively, or as SEQ ID NO: 7 and SEQ ID NO: 9, respectively, or as SEQ ID NO: 3 and SEQ ID NO: 11, respectively.

15. The polynucleotide molecule according to claim 13, wherein The nucleotide sequences of the polynucleotide molecule encoding the heavy chain variable region are shown as SEQ ID NO: 45, 46, or 47, respectively, and the nucleotide sequence encoding the light chain variable region is shown as SEQ ID NO:

44.

16. The polynucleotide molecule according to claim 13, wherein The nucleotide sequences of the polynucleotide molecule encoding the heavy chain variable region are shown as SEQ ID NO: 49, 50, or 51, respectively, and the nucleotide sequence encoding the light chain variable region is shown as SEQ ID NO:

52.

17. The polynucleotide molecule according to claim 13, wherein, The nucleotide sequences encoding the heavy chain variable regions of the polynucleotide molecules are shown as SEQ ID NO: 45, 46 or 47 respectively, and the nucleotide sequence encoding the light chain variable region is shown as SEQ ID NO:

48.

18. An expression vector, characterized in that, The expression vector contains the polynucleotide molecule as described in any one of claims 13-17.

19. A host cell, characterized in that, The host cell contains the expression vector as described in claim 18.

20. A method for preparing an antibody or an antigen-binding fragment thereof that binds to human IL-33 as described in any one of claims 1-12, characterized in that, The method comprises the following steps: a) Culturing the host cell as described in claim 19 under expression conditions to express the antibody or antigen-binding fragment thereof that binds to human IL-33; b) Separating and purifying the antibody or antigen-binding fragment thereof that binds to human IL-33 as described in a).

21. A composition, characterized in that, The composition contains the antibody or antigen-binding fragment thereof that binds to human IL-33 as described in any one of claims 1-12 and a pharmaceutically acceptable carrier.

22. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate contains: (a) An antibody moiety, which contains the antibody or antigen-binding fragment thereof that binds to human IL-33 as described in any one of claims 1-12; and (b) A conjugate moiety conjugated to the antibody moiety, and the conjugate moiety is selected from the group consisting of: a detectable label, a radionuclide, an enzyme, or a combination thereof.

23. Use of the antibody or antigen-binding fragment thereof that binds to human IL-33 as described in any one of claims 1-12, or the composition as described in claim 21, or the antibody-drug conjugate as described in claim 22 in the preparation of a medicament for treating asthma, arthritis, atopic / allergic dermatitis, chronic rhinosinusitis, chronic obstructive pulmonary disease (COPD), systemic sclerosis, liver fibrosis, psoriasis, ulcerative colitis, Crohn's disease, multiple sclerosis, diabetic kidney disease, psoriasis, eosinophilic esophagitis, diabetic macular edema, age-related macular degeneration, dry eye disease.

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