CN111620948B

CN111620948B - Antibodies to IL-33

Info

Publication number: CN111620948B
Application number: CN202010521408.9A
Authority: CN
Inventors: 钱峰; 王军; 段世鑫
Original assignee: Nanjing Saixin Biotechnology Co ltd
Current assignee: Nanjing Saixin Biotechnology Co ltd
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2022-04-08
Anticipated expiration: 2040-06-10
Also published as: CN111620948A

Abstract

The present invention provides a monoclonal antibody or binding fragment thereof, which non-fully humanized monoclonal antibody or fragment thereof specifically recognizes IL-33, comprising at least 1 variable region or mutated sequence thereof selected from the group consisting of: 1) comprises the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3, a heavy chain CDR region sequence; and/or, 2) comprises SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6, and a light chain CDR region sequence. The humanized monoclonal antibody or the binding fragment thereof provided by the invention has high affinity and strong stability.

Description

Antibodies to IL-33

Technical Field

The invention relates to the field of biotechnology medicine. In particular, the invention relates to antibodies capable of specifically recognizing and binding to human IL-33 molecules.

Background

IL-33 is a key member of the IL-1 family, which plays an important role in tissue and metabolic homeostasis, infection, inflammation, cancer, and neurological diseases. IL-33 binds with high affinity to interleukin 1 receptor 4(IL-1R 4; also known as oncostatin 2[ ST2]), and forms a ternary complex with IL-1RAcP to form a signaling complex. This signaling complex results in a series of myddosomes (with MyD88 and IRAK family members) dependent downstream signaling. IL-33 acts as an alarm, produced by damaged barrier layer cells (endothelial and epithelial cells). When cells (e.g., mast cells or basophils) are stimulated by IL-33, type 2 cytokines, such as IL-4, IL-5, and IL-13, are produced.

Preclinical studies have shown that IL-33 has a key role in many asthmatics and allergic diseases. Blocking of the IL-33 signaling pathway can be achieved by neutralizing antibodies to IL-33 or IL-1R4, genetic deletions of IL-33 or IL-1R4, or soluble fusion proteins of IL-1R4 coupled to Fc (Coyleetal, 1999, J.Exper.Med190(7): 895-902). In models of inflammation induced by allergens (e.g., dust mites, cockroaches, or alternaria (alternaria) fungi), IL-33 plays an important role in driving inflammation and airway remodeling, among others. In pharmacological models that rely on adjuvants, such as aluminum hydroxide (alum) or sodium urate crystals, IL-33 plays an important role in the sensitization phase of the model and in inducing the production of type 2 cytokines, such as IL-5 and IL-13. IL-33 has also been found to play an important role in the inflammatory response associated with airway viral infection. Damage to airway epithelium caused by viral infection can trigger the release of IL-33 and alter the type of immune response.

Various cytokines are involved in pathogenesis in diseases such as chronic rhinosinusitis with nasal polyps (CRSwNP), Atopic Dermatitis (AD) and asthma. The IL-33 receptor ST2 is expressed in many cell types associated with type 2 inflammation, including mast cells, basophils, Th2 cells, type 2 innate lymphoid cells. These cells produce a number of inflammatory cytokines upon stimulation by IL-33, particularly cytokines associated with type 2 inflammation, including IL-5, IL-13, IL-4, IL-31, and IL-9. Other cytokines and chemokines are also produced, which play an important role in the recruitment of other inflammatory cells. Initial release of IL-33 is triggered by epithelial damage to the body or mucosal surface. Disease-related triggers include allergens with proteolytic activity, physical damage to the epithelium, viruses, and fungi and bacteria common to body surfaces. In disease tissues rich in eosinophils and mast cells, damage to the epithelium initiates a cascade of reactions that release IL-33, which acts on local target cells and drives the production of a variety of cytokines that are important to the type 2 inflammatory response.

Disclosure of Invention

Humanized antibodies are antibodies from non-human species (e.g., murine antibodies) whose protein sequences have been modified to increase their similarity to antibodies in humans, which substantially retain the affinity and specificity of the original antibody, while also reducing heterogeneity. The humanization of murine antibodies is achieved by genetic engineering to achieve a profile as similar as possible to that of the human antibody molecules, thereby avoiding the recognition by the human immune system. Humanization is carried out following two basic principles, namely to maintain or improve the affinity and specificity of the antibody and also to reduce or eliminate the immunogenicity of the antibody. Currently humanized antibodies can be divided into four classes: human-murine chimeric antibodies, modified antibodies, resurfaced antibodies, and fully humanized antibodies. In recent years, humanized antibodies have shown good application prospects in tumor treatment, autoimmune diseases, cardiovascular diseases and other aspects.

In view of this, the present application provides humanized antibodies that recognize IL-33. The humanized antibodies selected according to the present application contain a number of specific "back mutations" in their variable region sequences, i.e., mutations from the humanized sequence back to the mouse sequence. These back mutations are important in maintaining the conformation and binding affinity of the original antibody, while they are performed on residues with the lowest incidence of potential T cell epitopes, thereby minimizing the risk of adverse immune responses to the antibody.

To generate a humanized antibody recognizing IL-33 with the desired and human framework having specific CDR region sequences, the inventors of the present application identified key residues in the human framework (outside the CDR region sequences) that affect CDR region display and designed a series of mutations of these key residues to restore the correct display of the CDR regions while minimizing the immunogenicity of the antibody and partially mutating the CDR region sequences of the humanized antibody to improve its affinity and specificity.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

in one aspect, the present application provides a monoclonal antibody or binding fragment thereof capable of specifically recognizing IL-33 comprising at least 1 variable region or mutated sequence thereof selected from the group consisting of:

1) comprises SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3, a heavy chain CDR region sequence; and/or the presence of a gas in the gas,

2) comprises SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6, and a light chain CDR region sequence.

In a specific embodiment of the present application, wherein the heavy chain CDR region sequence is in a framework sequence differing from SEQ ID NO: 47; or 1-10 amino acid residues of the framework sequence in which the light chain CDR region is located are different from SEQ ID NO: 34.

in a specific embodiment of the present application, wherein the framework sequence in which the heavy chain CDR region sequences are located differs from SEQ ID NO: 47.

in one embodiment of the present application, the heavy chain CDR region sequence is in a framework sequence having 1-5 amino acid residues different from seq id no: 47.

in one embodiment of the present application, the heavy chain CDR region sequence is in a framework sequence having 1-3 amino acid residues different from seq id no: 47.

in one embodiment of the present application, the heavy chain CDR region sequence is in a framework sequence differing in 3 amino acid residues from SEQ ID NO: 47.

in a specific embodiment of the present application, the light chain CDR regions are in a framework sequence that differs from SEQ ID NO: 34.

in one embodiment of the present application, the light chain CDR regions are in a framework sequence of 1-5 amino acid residues different from seq id no: 34.

in one embodiment of the present application, the light chain CDR regions are in a framework sequence of 1-4 amino acid residues different from seq id no: 34.

in one embodiment of the present application, the light chain CDR regions are in a framework sequence differing from the framework sequence of SEQ ID NO: 34.

in a specific embodiment of the present application, wherein the heavy chain CDR region sequence is in a framework sequence that differs from SEQ ID NO: 47: g044, S049, a075 and a 097.

In a specific embodiment of the present application, wherein the heavy chain CDR region sequence is in a framework sequence that differs from SEQ ID NO: 47: G044R, S049A, a075V and a 097T. In one embodiment of the present application, the heavy chain variable region sequence comprises the framework sequence set forth in seq id no: (1) SEQ ID NO: 10 or SEQ ID NO: 11; (2) SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15 or SEQ ID NO: 16; (3) SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. or SEQ ID NO: 20; and (4) SEQ ID NO: 21 or SEQ ID NO: 22.

in one embodiment of the present application, the framework sequence in which the heavy chain CDR region sequences are located is selected from the framework sequences represented by the following sequences: SEQ ID NO: 45. SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48. SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51 or SEQ ID NO: 52.

in a specific embodiment of the present application, the light chain CDR region sequence is in a framework sequence that differs from SEQ ID NO: 34: l004, G072 and T087.

In a specific embodiment of the present application, the light chain CDR region sequence is in a framework sequence that differs from SEQ ID NO: 34: L004V, G072R and T087V. In one embodiment of the present application, the light chain variable region comprises the framework sequence set forth in seq id no: (1) SEQ ID NO: 23. SEQ ID NO: 24 or SEQ ID NO: 25; (2) SEQ ID NO: 26; (3) SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29 or SEQ ID NO: 30, of a nitrogen-containing gas; and (4) SEQ ID NO: 31 or SEQ ID NO: 32.

in one embodiment of the present application, the light chain CDR region sequences are in a framework sequence selected from the group consisting of the framework sequences set forth in seq id nos: SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44 or SEQ ID NO: 53.

the present invention also provides a mutated sequence of the monoclonal antibody or binding fragment thereof.

In one embodiment of the invention, the mutation of the mutated sequence occurs in a light chain CDR region.

In a particular embodiment of the invention, the mutation is N034 and/or S035.

In a specific embodiment of the invention, the mutation is N034Q or N034S.

In one embodiment of the invention, the mutation is S035A.

In one embodiment of the invention, the heavy chain is selected from the group consisting of SEQ ID NO: 45. SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48. SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51. SEQ ID NO: 52.

in one embodiment of the invention, the light chain is selected from the group consisting of SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 53.

in one embodiment of the invention, the antibody or binding fragment thereof comprises SEQ ID NO: 45 and SEQ ID NO: 33, or a variant thereof comprising SEQ ID NO: 47 and SEQ ID NO: 33, or a variant thereof comprising SEQ ID NO: 45 and SEQ ID NO: 41, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 41, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 35, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 36, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 37, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 41, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 35, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 36, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 37, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 43, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 38, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 39, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 40, or which comprises SEQ ID NO: 46 and SEQ ID NO: 43, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 38, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 39, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 40, or which comprises SEQ ID NO: 47 and SEQ ID NO: 41, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 42, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 49 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 50 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 52 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 43, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 42, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 53, or a variant thereof comprising SEQ ID NO: 52 and SEQ ID NO: 53, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 58, or a sequence shown in fig.

In another aspect, the invention provides a polynucleotide encoding an antibody or binding fragment thereof as described above.

Another invention of the present invention provides an expression vector capable of expressing the antibody or binding fragment thereof described above, or the expression vector can contain the polynucleotide described above.

In another aspect, the present invention provides a host cell comprising the above-described expression vector, or a host cell having the above-described polynucleotide integrated into its genome.

Illustratively, the present invention has at least one of the following advantages:

the antibody or the binding fragment thereof provided by the invention has high affinity and high stability, has great potential medicinal value for preparing antibody medicines, and can treat, prevent or alleviate diseases related to IL-33, such as asthma, rheumatoid arthritis, septicemia, heart diseases, atherosclerosis, malignant tumors and the like.

Drawings

FIG. 1 is a graph showing the results of SDS-PAGE after purification of Protein A provided in the examples of the present invention.

FIG. 2 is a chart showing the results of SDS-PAGE experiments after SEC secondary purification provided in the examples of the present invention.

FIGS. 3-1 to 3-6 are graphs showing the results of experiments in which non-specific binding of antigens to the chip is detected by SPR. Wherein, fig. 3-1: the antibody is W3759-cAb1-xIgG4K.SP, the antigen is W3759-hPro1-WT, and the ratio is 1: 1; FIG. 3-2: the antibody is W3759-cAb1-z1-p1-uIgG4K.SP, the antigen is W3759-hPro1-WT, and the ratio is 1: 1; FIGS. 3-3: the antibody is W3759-cAb1-sz8-p1-uIgG4K.SP, the antigen is W3759-hPro1-WT, and the ratio is 1: 1; FIGS. 3-4: the antibody is W3759-cAb1-sz9-p1-uIgG4K.SP, the antigen is W3759-hPro1-WT, and the ratio is 1: 1; FIGS. 3 to 5: the antibody is W3759-cAb1-sz10-p1-uIgG4K.SP, the antigen is W3759-hPro1-WT, and the ratio is 1: 1; FIGS. 3 to 6: the antibody is W3759-cAb1-xIgG4K.SP, the antigen is W3759-hPro1-Mut, and the ratio is 1: 1.

FIG. 4-1 is a graph showing the results of an experiment in which the binding affinity of an antibody to an antigen is determined by the antigen coating method.

FIG. 4-2 is a graph showing the results of an experiment for detecting the affinity of an antibody for wild-type and mutant antigens by ELISA.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The present invention discloses monoclonal antibodies recognizing IL-33 that are almost fully humanized, thereby avoiding adverse immune responses to the antibody and are therefore safe for use in vivo. The antibodies of the present application are characterized by unique CDR region sequences and novel non-fully humanized framework sequences and designs, and partial mutations of the CDR region sequences that result in high affinity antibodies to the non-fully humanized framework sequences.

Description of the drawings: the variable regions of the antibodies provided herein comprise CDR regions and framework sequences, at least one residue in at least one of which is different from the corresponding fully human framework sequence.

The antibodies of the invention are molecules comprising at least the antigen-binding portion of an antibody, including in particular intact antibodies, such as polyclonal or monoclonal antibodies, as well as proteolytic fragments thereof, such as Fab or F (ab')2 fragments. Single chain antibodies are also within the scope of the invention.

The term "amino acid residue mutation" as used herein refers to substitution, insertion or deletion of a single amino acid residue. The term "back-mutation" as used herein refers to the substitution of a single amino acid residue found in the human antibody backbone to the corresponding amino acid residue found in the murine antibody backbone.

In the invention, W3759-hPro1-WT I is human IL-33, and W3759-hPro1-Mut is human IL-33 with epitope mutation.

The reference fully humanized heavy chain sequence to which back mutations were introduced was SEQ ID NO: 47; the fully humanized light chain sequence is SEQ ID NO: 34.

example 1 design of CDR region grafted antibody variable region sequences

1.1 determination of antibody sequence numbering and complementarity determining regions (CDR regions)

W3759-cAb1 (murine sequence) was automatically numbered (Dunbar and deane 2015) according to the Kabat numbering method by ANARCI software. The CDR regions in the W3759-cAb1 antibody sequence were determined by definition on the Antibodies website (see Table 1) (Martin2018, http:// www.bioinf.org.uk/abs/index. html # cdrid).

Table 1 dr. martin website definition of CDR

1.2 design and plasmid construction of humanization and post-translational modification removal

The sequences of murine antibody W3759-cAb1 (heavy chain SEQ ID NO: 45 and light chain SEQ ID NO: 33) were aligned to the IMGT human germline sequence library (Lefranc 2003) and analyzed to identify amino acids in the V-region backbone that may be important for the binding characteristics of the antibody. These amino acids were used to incorporate one or more variant CDR grafted antibodies, based on which a humanized germline sequence with the least difference in amino acid at the corresponding position in the alignment with the W3759-cAb1 sequence was selected as the humanization template and by the above identification, back-mutations were made to residues that may be critical for restoring the binding potency of the murine antibody, thereby designing a series of heavy and light chain V regions and constructing different humanized antibodies.

During the design of the back-mutation, the W3759-cAb1 sequence was also analyzed for the presence of the following sites: asparagine deamination sites (Asn-Gly or Asn-Ser) in CDR regions, aspartate isomerization sites (Asp-Gly) in CDR regions, unpaired cysteines in CDR regions or Framework (Framework) regions, and N-linked glycosylation sites (Asn-Xxx-Ser/Thr, where Xxx can be other amino acids besides proline) in CDR regions or Framework regions. Point mutations were designed empirically to remove this site. These mutations will also be introduced into the humanized antibody. The number of back mutations was determined by the initial murine sequence.

Plasmids bearing chimeric antibody sequences were synthesized by jingezhi: based on pcDNA3.1 plasmid, the plasmid containing antibody heavy chain variable region consists of the heavy chain variable region of W3759-cAb1, fused human IgG1 heavy chain CH1, hinge region, CH2 and CH3 sequences; a plasmid containing the variable region of the antibody light chain, which is composed of the variable region of the light chain of W3759-cAb1 fused with the sequence of the human Ig kappa CK domain. Each plasmid construct contains the same constant region sequence, only in the heavy chain or light chain variable region of the antibody difference.

1.3 Small transient expression and SDS-PAGE gel electrophoresis

First, 1mL of 1.2X10 was prepared⁶And a viability rate of greater than 95% of HEK293F cells for each antibody expression. Then, the plasmid DNA was mixed at a final concentration of 1. mu.g/mL with Lipofectamine at a final concentration of 2.5. mu.L/mL^TM2000 transfection reagents were mixed and added to the prepared cells. The transfected cells were placed in a 37 ℃ constant temperature shaker at 150rpm with 8% CO₂Culturing is carried out in a concentration environment.

After 5 days of culture, cells were removed by centrifugation at 4000rpm for 10 minutes at 25 ℃. After collecting the supernatant for k_offAnd (5) detecting and performing gel electrophoresis. Using NuPAGE^TM4-12% Bis-Tris protein gel (ThermoFisher) was electrophoresed in a loading of 13. mu.L using a PageRuler^TMProtein standards indicate sample band molecular weights.

1.4K_offSequencing (capture antibody method) (SPR method)

(1) Coating quilt

The activator was prepared by mixing 400mM EDC with 100mM NHS (GE) before the start of the experiment. The sensor chip was activated using an activator at a flow rate of 10. mu.L/min for 420 s. Then, anti-human Fc IgG (Jackson) diluted with 10mM NaAc (pH 4.5) at a final concentration of 30. mu.g/mL was injected into each of the 1-8 channels at a flow rate of 10. mu.L/min for 420 s. Finally, the chip was closed with 1M ethanomine-HCl (GE) for 420s at a flow rate of 10. mu.L/min.

(2) Capture antibodies and affinity assays

Blank supernatant control and antibody-containing supernatant were injected into the Fc1 and Fc2 channels, respectively, at a flow rate of 10 μ L/min for 50 s. 2M W3759-hPro1 and buffer were then injected into Fc1-Fc2 at a flow rate of 30. mu.L/min, bound for 300s, and subsequently dissociated for 2400 s. When the dissociation process is over, 10mM Glycine pH 1.5 as regeneration buffer injection channel.

(3) Regeneration

The chip was regenerated using 10mM Glycine, pH 1.5 buffer.

(4) Data analysis

The final binding dissociation curve is the result of subtracting the reference channel and the buffer channel. The data were fitted according to a 1:1 binding model.

1.5 antibody production

Plasmids containing the heavy and light chain variable region genes were co-expressed in Expi293F cells. Cells were harvested after 6 days of culture, supernatants were collected and protein purified using MabSelect SuRe equilibrated with wash buffer (100mM Tris, pH 7.0). The supernatant from the cell culture fluid was applied to the column through a sample injection valve. Then washing with 30 times column volume of washing buffer solution to remove impurities, and gradient-eluting with buffer solution B (0.1M Glycine pH 3.5) to obtain the target protein. A280 was then detected using Nanodrop 2000 and the protein concentration in the eluate was obtained from the formula A280/extinction coefficient. Finally, the purified antibody was subjected to SDS-PAGE gel electrophoresis and HPLC-SEC detection, and frozen at-80 ℃.

1.6 measurement of KD value (antigen-coating method) affinity by SPR

(1) Buffer replacement

W3759-hPro1-WT and W3759-hPro1-Mut were displaced into 1 XHBS-EP + buffer using a desalting column.

(2) Coating quilt

The activator was prepared by mixing 400mM MDC with 100mM NHS (GE) before the start of the experiment. The sensor chip was activated using an activator at a flow rate of 10. mu.L/min for 420 s. Then, 5. mu.g/mL W3759-hPro1-WT diluted with 10mM NaAcpH4.5 was injected into channels 1-6 at a flow rate of 10. mu.L/min for 30 s. mu.g/mLW 3759-hPro1-Mut diluted with 10mM NApH4.5 was injected into channel 7 at a flow rate of 10. mu.L/min for 30 s. Finally, the chip was closed with 1M ethanomine-HCl (GE) for 420s at a flow rate of 10. mu.L/min.

(3) Detection of

Different concentrations of antibody and buffer were injected into the Fc1 and Fc2 channels at a flow rate of 30 μ L/min for 3600s of binding, followed by dissociation for 3600 s. After each cycle of interaction detection, the sensor chip surface was regenerated using 10mM Glycine pH 1.5 at a flow rate of 10. mu.L/min for 30 s.

(4) Regeneration

The chip was regenerated using 10mM Glycine, pH 1.5 buffer.

(5) Data analysis

1.7ELISA determination of EC₅₀(effective half)

W3759-hPro1(IL-33) was coated on ELISA plates at 0.125. mu.g/mL overnight at 4 ℃. After blocking wash plates, the antibody was diluted in half using blocking solution and incubated at room temperature for 2 h. Plates were then washed and secondary antibodies (Goat anti Human IgG-Fc-HRP, Bethy-A80-304P) were incubated for 1 h. After washing the plates, TMB substrate was added and the reaction was stopped by addition of 2M HCl. Finally, the absorbance was read at 450nm using a microplate reader.

2 results

2.1 sequence analysis

The amino acid sequence of the variable region of the W3759-cAb1 antibody is shown below. CDR regions (single underlined and bolded) were determined according to the Kabat CDR region definition method. The point mutation sites are highlighted by double underlining italics.

W3759-cAb1_VH：(SEQ ID NO:45)

W3759-cAb1_VL：(SEQ ID NO:33)

2.2 humanization design and plasmid construction

Finding out the human embryonic line gene with the highest similarity with the W3759-cAb1 antibody light chain variable region framework sequence through database alignment: hIGKV7-3 + hIGKJ 2-03 human germline genes (Table 2, 3). The germline gene of human origin with the highest similarity to the framework sequence of the heavy chain variable region of the W3759-cAb1 antibody was hIGHV3-21 + hIGHJ 1+ 01 (Table 2, 3). To maximize the ability to maintain antigen-antibody binding, we designed empirically two different sets of humanized antibodies by back-mutation. The first group comprised 6 humanized heavy and 6 light chain variable region variants (point mutation sites included in the light chain variants). The second group comprised 2 humanized heavy chain and 7 light chain variable region variants. These mutations maintain the CDR regions in their original conformation and retain their affinity for the antigen if they bind to the antigen.

The humanized variable region sequence was reverse translated from kasugar, codon optimized and gene synthesized.

TABLE 2 humanized design of variable region genes

TABLE 3 humanized design of variable region genes

Description of the drawings: ".." means that the amino acid at the corresponding position is the same as the amino acid at it.

2.3 transient transfection of chimeric and humanized antibodies

The 23 strains of antibodies were expressed using eukaryotic expression plasmids pcDNA3.1 and 293F cells (tables 4 and 5), including 1 chimeric antibody, 2 hybrid antibodies and 27 humanized and post-translationally modified mutant antibodies. K-Generation Using antibody-expressing cell culture supernatants_offAnd (5) sequencing experiments.

TABLE 4 humanized antibody design one

TABLE 5 humanized antibody design II

2.4K_offRank analysis

K-Generation Using antibody-expressing cell culture supernatants_offRanking experiments (analysis of results see table 6). Since antibody dissociation is very slow, the kd value measured is less than the detection limit of Biacore (1E-05), and therefore the kd value cannot be used to compare the binding capacity of antigen antibodies. Also, due to the lack of curvature of the binding curve, an accurate ka value cannot be obtained. Finally, the ka value of the antibody is indicated by the intensity of the expected binding response.

TABLE 6SPR data analysis

As can be seen from Table 6SPR, the binding activity of the antibody to IL-33 was not consistent after the modification. The antibodies with the Expected binding higher than W3759-cAb1-xIgG4K.SP include W3759-cAb1-sz6-xIgG4K.SP, W3759-cAb1-sz8-xIgG4K.SP, W3759-cAb1-sz9-xIgG4K.SP, W3759-cAb1-sz10-xIgG4K.SP, W3759-cAb1-sz 11-xIgG4K.SP. Among them, the best binding activity was W3759-cAb1-sz 9-xIgG4K.SP.

2.5ELISA sequencing assay

Due to the nature of the antibody, the constructed humanized antibodies cannot be compared with the exact kd values obtained. Therefore, these antibodies were simultaneously analyzed for their ability to bind to the antigen using ELISA assays. The results are shown in Table 7-1, Table 7-2 and Table 7-3.

TABLE 7-1 analysis of ELISA data 1

TABLE 7-2 analysis of ELISA data 2

Tables 7-3 analysis of ELISA data 3

As can be seen from the ELISA data in tables 7-1, 7-2 and 7-3, the binding activity of the engineered antibody to IL-33 was varied, and both the negative control and human IgG4 did not bind to IL-33. The three engineered antibodies with the best binding activity were W3759-cAb1-sz8-xIgG4K.SP, W3759-cAb1-sz9-xIgG4K.SP and W3759-cAb1-sz10-xIgG4K.SP, respectively, in which the EC measured for W3759-cAb1-sz9-xIgG4K.SP and W3759-cAb1-sz10-xIgG4K.SP and W3759-cAb1-xIgG4K.SP₅₀Substantially identical.

2.6 expression and purification of antibodies

By analyzing the SPR and ELISA data together, W3759-cAb1-z1-uIgG4K.SP, W3759-cAb1-sz8-xIgG4K.SP, W3759-cAb1-sz9-xIgG4K.SP, W3759-cAb1-sz10-xIgG4K.SP were selected as candidate antibodies, and N034Q mutations were introduced into humanized antibodies, plus 1 chimeric antibody, and expressed using eukaryotic expression plasmid pcDNA3.1 and 293F cells. Cell culture supernatants expressing the antibodies were collected and purified using ProteinA chromatography columns, and W3759-cAb1-sz8-xIgG4K.SP, W3759-cAb1-sz9-xIgG4K.SP were purified in two steps to a purity of 90% or more. The experimental results after the purification of ProteinA are shown in FIG. 1; the experimental results after SEC purification are shown in fig. 2; the experimental results of SEC-HPLC are shown in FIGS. 3-1 to 3-5.

The numbering shown in FIG. 1 and the results of the experiments are shown below.

No.	Protein Name	MW(kDa)	Concentration(mg/ml)	Buffer	Purity(％)
						1	W3759-cAb1-xlgG4K.SP	145.1/25+50	3.91	PBS	97.36％
2	W3759-cAb1-z1-p1-ulgG4K.SP	144.5/25+50	4.18	PBS	98.48％
						3	W3759-cAb1-sz8-p1-xlgG4K.SP	144.8/25+50	1.17	PBS	88.38％
4	W3759-cAb1-sz9-p1-xlgG4K.SP	144.6/25+50	1.81	PBS	73.98％
						5	W3759-cAb1-sz10-p1-xlgG4K.SP	144.8/25+50	3.34	PBS	96.28％

As can be seen from FIG. 1, after the ProteinA is purified, the bands of W3759-cAb1-xIgG4K.SP, W3759-cAb1-z1-p1-uIgG4K.SP and W3759-cAb1-sz10-p1-xIgG4K.SP after one-step purification are single (the antibody is one band in a non-reduction state, and is divided into a heavy chain band and a light chain band after reduction), and the purity is higher; and W3759-cAb1-sz8-p1-xIgG4K.SP and W3759-cAb1-sz9-p1-xIgG4K.SP have hybrid bands and hybrid proteins after further purification, and further purification is needed. Further purification of W3759-cAb1-sz8-p1-xIgG4K.SP and W3759-cAb1-sz9-p1-xIgG4K.SP are shown in FIG. 2.

The numbering shown in FIG. 2 and the results of the experiment are shown below.

No.	Protein Name	MW(kDa)	Concentration(mg/ml)	Buffer	Purity(％)
						3	W3759-cAb1-sz8-p1-xlgG4K.SP	144.8/25+50	0.88	PBS	98.52％
4	W3759-cAb1-sz9-p1-xlgG4K.SP	144.6/25+50	1.05	PBS	99.62％

As can be seen from FIG. 2, the bands of the W3759-cAb1-sz8-p1-xIgG4K.SP and the W3759-cAb1-sz9-p1-xIgG4K.SP after two-step purification are single and have high purity.

The purity of the purified antibody was determined by HPLC as follows: the purity of W3759-cAb1-xlgG4K.SP can reach 97.36 percent; the purity of W3759-cAb1-z1-p1-ulgG4K.SP can reach 98.48 percent; the purity of W3759-cAb1-sz10-p1-xlgG4K.SP can reach 96.28 percent; the purity of W3759-cAb1-sz8-p1-xlgG4K.SP can reach 98.52 percent; the purity of W3759-cAb1-sz9-p1-xlgG4K.SP can reach 99.62 percent, the number of hetero peaks is few, and the purity of the five proteins after purification can reach the experimental requirements.

2.7SPR detection (antigen coating method)

Since the lack of curvature of the binding curve obtained by the coated antibody method in SPR detection does not allow accurate ka measurement, attempts have been made to increase the concentration of the antigen passing through. However, by measuring the signal of different concentrations of antigen flowing through the reference channel, it was found that the antigen showed an increasing non-specific interaction with the chip with increasing concentration, making it impossible to measure true kinetic parameters under such conditions. Therefore, it is considered that the detection is carried out by the antigen coating method (the analysis results are shown in Table 8 and FIGS. 3-1 to 3-6).

TABLE 8 test results by the coated antigen method

FIGS. 3-1 to 3-6 are graphs showing the affinity of the antibody to the antigen measured by the antigen coating method. FIGS. 3-1 to 3-5 show that after IL-33 antigen is coated, the antibody and antigen affinity curves obtained by using W3759-cAb1, W3759-cAb1-z1-p1-uIgG4K.SP, W3759-cAb1-sz8-p1-xIgG4K.SP, W3759-cAb1-sz9-p1-xIgG4K.SP, and W3759-cAb1-sz10-p1-xIgG4K.SP as mobile phases show that W3759-cAb1-sz10-p 1-xIgGK.SP has a stronger antigen binding ability than W3759-cAb 1. FIGS. 3-6 are affinity curves of antibody and antigen obtained by coating IL-33 mutant and using W3759-cAb1 as mobile phase, which shows that neither mutant antigen nor modified antibody nor chimeric antibody is combined.

As shown in fig. 3-1 to fig. 3-6 and table 8, although the method cannot measure true affinity due to the bivalent effect, the KD values measured by the method are still suitable for comparing strong and weak trends of affinity among antibodies. And finally, determining that the affinity of the modified antibody and the antigen is 77.6pM at the strongest and 15.7nM at the weakest, and the modified antibody has no affinity with the mutant antigen. Among them, W3759-cAb1-sz10-p1-xIgG4K.SP has a stronger antigen-binding ability than that of the chimeric antibody (0.21 nM).

2.8ELISA detection

To further verify the analytical results of SPR, the affinity of the engineered antibody to the antigen was determined using ELISA and the results are shown in Table 9, FIGS. 4-1 and 4-2.

TABLE 9 measurement of the results of the ELISA method

FIGS. 4-1 and 4-2 are a graph of the affinity of the engineered antibodies to IL-33 and IL-33 mutants determined using ELISA. As can be seen from FIG. 4-1, compared with human IgG4, W3759-cAb1, W3759-cAb1-z1-p1-uIgG4K.SP, W3759-cAb1-sz8-p1-xIgG4K.SP, W3759-cAb1-sz9-p1-xIgG4K.SP, W3759-cAb1-sz10-p1-xIgG4K.SP5 antibodies all have certain affinity with IL-33, and the engineered antibodies W3759-cAb1-sz10-p 1-xG4K.SP and the chimeric antibody W3759-cAb1 have higher affinity with IL-33.

As is clear from FIGS. 4-2, at concentrations of 10nM or less, the antibodies W3759-cAb1, W3759-cAb1-z1-p1-uIgG4K.SP, W3759-cAb1-sz8-p1-xIgG4K.SP, W3759-cAb1-sz9-p1-xIgG4K.SP, and W3759-cAb1-sz10-p1-xIgG4K.SP5 did not substantially bind to the IL-33 mutant, and non-specific binding occurred at high concentrations.

According to the analysis of FIG. 4-1, FIG. 4-2 and Table 9, the results obtained by ELISA and the results obtained by SPR have better consistency, the consistency of W3759-cAb1-sz10-p1-xIgG4K.SP and chimeric antibody is higher, the effect of W3759-cAb1-sz8-p1-xIgG4K.SP is better, and in ELISA, compared with other modified antibodies, the modified antibodies have higher affinity with antigens.

The antibody or the binding fragment thereof provided by the invention has high affinity and high stability, has great potential medicinal value for preparing antibody medicines, and provides a core basis for developing medicines for treating, preventing or relieving IL-33 related diseases, such as asthma, rheumatoid arthritis, septicemia, heart diseases, atherosclerosis and malignant tumors.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

Sequence listing

<110> Nanjing Sai New Biotechnology Co., Ltd

<120> antibodies against IL-33

<160> 53

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR1

<400> 1

Gly Phe Thr Phe Asn Asp Tyr Gly Met Ser

1 5 10

<210> 2

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR2

<400> 2

Ser Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210> 3

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR3

<400> 3

Leu Pro Ala Pro Phe Val Ser

1 5

<210> 4

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR1

<400> 4

Arg Ala Ser Glu Ser Val Asp Arg Tyr Gly Asn Ser Phe Met His

1 5 10 15

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR2

<400> 5

Arg Ala Ser Asn Leu Asp Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR3

<400> 6

Gln Gln Ser Asn Glu Asp Pro Leu Thr

1 5

<210> 7

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> NQ

<400> 7

Arg Ala Ser Glu Ser Val Asp Arg Tyr Gly Gln Ser Phe Met His

1 5 10 15

<210> 8

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> NS

<400> 8

Arg Ala Ser Glu Ser Val Asp Arg Tyr Gly Ser Ser Phe Met His

1 5 10 15

<210> 9

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SA

<400> 9

Arg Ala Ser Glu Ser Val Asp Arg Tyr Gly Asn Ala Phe Met His

1 5 10 15

<210> 10

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H1FR1

<400> 10

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser

20 25

<210> 11

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H2FR1

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 12

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H1FR2

<400> 12

Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala

1 5 10

<210> 13

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H2FR2

<400> 13

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

1 5 10

<210> 14

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H3FR2

<400> 14

Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val Ser

1 5 10

<210> 15

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H4FR2

<400> 15

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

1 5 10

<210> 16

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H5FR2

<400> 16

Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val Ala

1 5 10

<210> 17

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H1FR3

<400> 17

Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Thr Arg

20 25 30

<210> 18

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H2FR3

<400> 18

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 19

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H3FR3

<400> 19

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg

20 25 30

<210> 20

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H4FR3

<400> 20

Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Ser Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg

20 25 30

<210> 21

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H1FR4

<400> 21

Trp Gly Gln Gly Thr Leu Val Lys Val Ser Ala

1 5 10

<210> 22

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H2FR4

<400> 22

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 23

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> L1FR1

<400> 23

Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys

20

<210> 24

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> L2FR1

<400> 24

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys

20

<210> 25

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> L3FR1

<400> 25

Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys

20

<210> 26

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> L1FR2

<400> 26

Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210> 27

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> L1FR3

<400> 27

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys

20 25 30

<210> 28

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> L2FR3

<400> 28

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys

20 25 30

<210> 29

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> L3FR3

<400> 29

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys

20 25 30

<210> 30

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> L4FR3

<400> 30

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys

20 25 30

<210> 31

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> L1FR4

<400> 31

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

1 5 10

<210> 32

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> L2FR4

<400> 32

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 33

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-mVK

<400> 33

Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 34

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-hVK

<400> 34

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 35

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-zVK1-p1

<400> 35

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Gln Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 36

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-zVK1-p2

<400> 36

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Ser Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 37

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-zVK1-p3

<400> 37

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Asn Ala Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 38

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-zVK2-p1

<400> 38

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Gln Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 39

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-zVK2-p2

<400> 39

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Ser Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 40

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-zVK2-p3

<400> 40

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Asn Ala Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 41

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-szVK1

<400> 41

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 42

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-szVK2

<400> 42

Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 43

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-szVK3

<400> 43

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 44

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-szVK4

<400> 44

Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 45

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-mVH

<400> 45

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Thr Arg Leu Pro Ala Pro Phe Val Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Lys Val Ser Ala

115

<210> 46

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-zVH3

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Leu Pro Ala Pro Phe Val Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 47

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-szVH1

<400> 47

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Pro Ala Pro Phe Val Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 48

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-szVH2

<400> 48

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Leu Pro Ala Pro Phe Val Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 49

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-szVH3

<400> 49

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Leu Pro Ala Pro Phe Val Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 50

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-szVH4

<400> 50

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Leu Pro Ala Pro Phe Val Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 51

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-szVH5

<400> 51

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Leu Pro Ala Pro Phe Val Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 52

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-szVH6

<400> 52

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Pro Ala Pro Phe Val Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 53

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> W3759-zVK3

<400> 53

Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Gln Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that specifically recognizes IL-33,

(1) a heavy chain variable region sequentially comprising heavy chain CDR1-3 region sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3; and the combination of (a) and (b),

(2) a light chain variable region sequentially comprising light chain CDR1-3 region sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6;

the framework sequence of the heavy chain CDR region sequence has 1-10 amino acid residues different from SEQ ID NO. 47; the light chain CDR region is located in the framework sequence of 1-5 amino acid residues different from SEQ ID NO. 34.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain CDR region sequence is in a framework sequence differing from SEQ ID No.47 in 1-5 amino acid residues; the light chain CDR region is located in the framework sequence of 1-3 amino acid residues different from SEQ ID NO. 34.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain CDR region sequence is in a framework sequence differing from SEQ ID No.47 in 1-3 amino acid residues.

4. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain CDR region sequence is in a framework sequence that differs from SEQ ID No.47 at least one residue selected from the group consisting of: g044, S049, a075 and a 097.

5. The monoclonal antibody or antigen-binding fragment thereof of claim 4, wherein said SEQ ID No.47 is mutated: G044R, S049A, a07V5 and a 097T.

6. The monoclonal antibody, or antigen-binding fragment thereof, of claim 1, wherein the heavy chain variable region sequence comprises the framework sequences set forth in seq id no:

(1) SEQ ID NO.10 or SEQ ID NO. 11;

(2) SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 or SEQ ID NO. 16;

(3) SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, or SEQ ID No. 20; and

(4) SEQ ID NO.21 or SEQ ID NO. 22.

7. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain CDR region sequences are in a framework sequence that differs from SEQ ID No.34 at least one residue selected from the group consisting of: l004, G072 and T087.

8. The monoclonal antibody or antigen-binding fragment thereof of claim 7, wherein said SEQ ID No.34 is mutated: L004V, G072R and T087V.

9. The monoclonal antibody, or antigen-binding fragment thereof, of claim 1, wherein the light chain variable region sequence comprises the framework sequences set forth in seq id no:

(1) SEQ ID No.23, SEQ ID No.24 or SEQ ID No. 25;

(2)SEQ ID NO.26；

(3) SEQ ID No.27, SEQ ID No.28, SEQ ID No.29 or SEQ ID No. 30; and

(4) SEQ ID NO.31 or SEQ ID NO. 32.

10. The monoclonal antibody or antigen binding fragment thereof of claim 1, wherein the heavy chain is selected from the group consisting of SEQ ID No.45, SEQ ID No.46, SEQ ID No.47, SEQ ID No.48, SEQ ID No.49, SEQ ID No.50, SEQ ID No.51, SEQ ID No. 52; the light chain is selected from the group consisting of SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO. 53.

11. The monoclonal antibody or antigen binding fragment thereof of claim 10, wherein SEQ ID No.35, SEQ ID No.38 or SEQ ID No.53 are mutated: N034Q.

12. The monoclonal antibody or antigen binding fragment thereof as claimed in claim 1, which comprises SEQ ID No.45 and SEQ ID No.33, or which comprises SEQ ID No.47 and SEQ ID No.33, or which comprises SEQ ID No.45 and SEQ ID No.41, or which comprises SEQ ID No.48 and SEQ ID No.35, or which comprises SEQ ID No.48 and SEQ ID No.36, or which comprises SEQ ID No.48 and SEQ ID No.37, or which comprises SEQ ID No.46 and SEQ ID No.41, or which comprises SEQ ID No.46 and SEQ ID No.35, or which comprises SEQ ID No.46 and SEQ ID No.36, or which comprises SEQ ID No.46 and SEQ ID No.37, or which comprises SEQ ID No.48 and SEQ ID No.43, or which comprises SEQ ID No.48 and SEQ ID No.38, or which comprises SEQ ID No.48 and SEQ ID No.48, or SEQ ID No.39, or which SEQ ID No.40, or which comprises SEQ ID NO.46 and SEQ ID NO.43, or which comprises SEQ ID NO.46 and SEQ ID NO.38, or which comprises SEQ ID NO.46 and SEQ ID NO.39, or which comprises SEQ ID NO.46 and SEQ ID NO.40, or which comprises SEQ ID NO.47 and SEQ ID NO.41, or which comprises SEQ ID NO.48 and SEQ ID NO.42, or which comprises SEQ ID NO.48 and SEQ ID NO.44, or which comprises SEQ ID NO.49 and SEQ ID NO.44, or which comprises SEQ ID NO.50 and SEQ ID NO.44, or which comprises SEQ ID NO.51 and SEQ ID NO.44, or which comprises SEQ ID NO.52 and SEQ ID NO.44, or which comprises SEQ ID NO.51 and SEQ ID NO.43, or which comprises SEQ ID NO.51 and SEQ ID NO.42, or which comprises SEQ ID NO.51 and SEQ ID NO.53, or it comprises SEQ ID NO.52 and SEQ ID NO.53, or it comprises the sequences shown in SEQ ID NO.51 and SEQ ID NO. 58.

13. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-12.

14. An expression vector capable of expressing the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-12, or comprising the polynucleotide of claim 13.

15. A host cell comprising the expression vector of claim 14, or having the polynucleotide of claim 13 integrated into its genome.