CN113493513B

CN113493513B - Anti-human IL-33 neutralizing autoantibody and preparation method and application thereof

Info

Publication number: CN113493513B
Application number: CN202110740256.6A
Authority: CN
Inventors: 徐大模; 刘志刚; 黄啸谷
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2022-07-12
Anticipated expiration: 2041-06-30
Also published as: CN113493513A

Abstract

The application provides an anti-human IL-33 neutralizing autoantibody, wherein a heavy chain variable region of the anti-human IL-33 neutralizing autoantibody comprises a heavy chain variable region as shown in SEQ ID NO:1 and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO:2, or a pharmaceutically acceptable salt thereof. The anti-human IL-33 neutralizing autoantibody can bind and neutralize IL-33 activity, and specifically block IL-33 mediated biological functions. The application also provides a preparation method and application of the anti-human IL-33 neutralizing autoantibody.

Description

Anti-human IL-33 neutralizing autoantibody and preparation method and application thereof

Technical Field

The application relates to the field of biomedicine, in particular to an anti-human IL-33 neutralizing autoantibody and a preparation method and application thereof.

Background

Allergic asthma is the main clinical phenotype of bronchial asthma, is a common chronic airway inflammatory disease, slightly affects the work and life of patients, and can cause residual diseases and even death [1 ]. The incidence of allergic asthma has been rising worldwide for over thirty years. Currently, about 3 billion allergic asthma patients worldwide, and this figure is expected to exceed 4 billion by 2025. Therefore, how to effectively control the occurrence and development of allergic asthma, particularly severe asthma, has become a great global challenge in public health, clinical medicine and economy.

Interleukin-33 (IL-33) is a multifunctional immune activator, which can be induced by various factors, including environmental factors such as allergen, inflammatory molecules and pathogen infection, and is closely related to various diseases, including infection and autoimmunity, especially allergic diseases, such as allergic rhinitis, dermatitis, conjunctivitis, food allergy and anaphylactic shock [2-5 ]. Unlike other cytokines, IL-33 protein is produced and secreted by cells, and is also stored in the nucleus and released when inflammation and necrosis occur in the cells. The research finds that the IL-33 released by secretion can directly activate pathological innate and adaptive immune cells through a receptor ST2 on a cell membrane to cause or aggravate autoimmune and allergic diseases; in allergic asthma, IL-33 directly activates pathological ILC2, macrophage, eosinophil, mast and Th2 cells in asthma and induces a variety of important cytokines including IL-4, IL-5, IL-13, TNF α to trigger allergic and partially non-allergic asthmatic responses [2-5 ]. Therefore, IL-33 may be an important diagnostic, prognostic and therapeutic new target for asthma.

In order to treat severe asthma, many humanized monoclonal antibody drugs against the above cytokines have been developed or are under development, show a certain therapeutic effect, and are not ideal; at the same time, no antibody medicine targeting IL-33 is clinically applied at home and abroad, and the importance of the antibody medicine in the treatment of asthma and other diseases is unknown.

Disclosure of Invention

In view of the above, the present application provides an anti-human IL-33 neutralizing autoantibody, which can bind to and specifically block IL-33 mediated biological functions, and has a wide application prospect in the preparation of products for diagnosing, preventing, treating or prognostically evaluating IL-33 related diseases.

In a first aspect, the present application provides an anti-human IL-33 neutralizing autoantibody, wherein the heavy chain variable region of said anti-human IL-33 neutralizing autoantibody comprises the amino acid sequence set forth in SEQ ID NO:1 and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

Optionally, the anti-human IL-33 neutralizing autoantibody further comprises a heavy chain constant region that is hIgG1, hIgG2, hIgG3, or hIgG 4.

Alternatively, the heavy chain of the anti-human IL-33 neutralizing autoantibody comprises the amino acid sequence set forth in SEQ ID NO: 3.

Optionally, the light chain of the anti-human IL-33 neutralizing autoantibody comprises the amino acid sequence as set forth in SEQ ID NO: 4.

The application provides an anti-human IL-33 neutralizing autoantibody, which can be induced to generate in a patient and has wide application prospect in products for diagnosis, prevention, treatment or prognosis evaluation of IL-33 mediated related diseases as a neutralizing antibody.

In a second aspect, the present application provides a method for preparing the anti-human IL-33 neutralizing autoantibody of the first aspect, comprising obtaining the neutralizing autoantibody by genetic engineering or in vitro induction.

The second aspect of the present application provides a simple and convenient method for preparing the anti-human IL-33 neutralizing autoantibody, and the anti-human IL-33 neutralizing autoantibody with strong activity is prepared.

In a third aspect, the present application provides a nucleic acid molecule encoding an anti-human IL-33 neutralizing autoantibody according to the first aspect.

In a fourth aspect, the present application provides an expression cassette, a recombinant vector or a recombinant cell comprising the nucleic acid molecule of the third aspect.

In a fifth aspect, the present application provides a pharmaceutical composition comprising an anti-human IL-33 neutralizing autoantibody according to the first aspect.

In a sixth aspect, the present application provides the use of the anti-human IL-33 neutralizing autoantibody of the first aspect, the anti-human IL-33 neutralizing autoantibody produced by the production method of the second aspect, the nucleic acid molecule of the third aspect, the expression cassette, the recombinant vector or the recombinant cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect in a reagent for the diagnosis or prognosis of an IL-33 mediated disease.

In a seventh aspect, the present application provides a use of the anti-human IL-33 neutralizing autoantibody of the first aspect, the anti-human IL-33 neutralizing autoantibody produced by the production method of the second aspect, the nucleic acid molecule of the third aspect, the expression cassette, the recombinant vector or the recombinant cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect in the manufacture of a medicament for preventing or treating an IL-33 mediated disease.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the results of in vitro detection of neutralizing autoantibodies against human IL-33 induced production, wherein A in FIG. 1 is the result of detection of polypeptide antigen 1 added with human IL-33, B in FIG. 1 is the result of detection of polypeptide antigen 2 added with human IL-33, and C in FIG. 1 is the result of detection of polypeptide antigen 3 added with human IL-33.

FIG. 2 is a graph showing the results of measurement of anti-cytokine autoantibody titer in allergic asthma patients, wherein A in FIG. 2 is the result of measurement of anti-IL-4 antibody in mild allergic asthma patients, B in FIG. 2 is the result of measurement of anti-IL-5 antibody in mild allergic asthma patients, C in FIG. 2 is the result of measurement of anti-IL-13 antibody in mild allergic asthma patients, D in FIG. 2 is the result of measurement of anti-IL-33 antibody in mild allergic asthma patients, E in FIG. 2 is the result of measurement of anti-IL-33 antibody in severe allergic asthma patients, and F in FIG. 2 is the positive rate of anti-human IL-33 autoantibody in mild and severe allergic asthma patients.

FIG. 3 is a graph showing the results of type detection of anti-human IL-33 neutralizing autoantibodies in patients with mild or severe allergic asthma, wherein A in FIG. 3 is an IgG type antibody, B in FIG. 3 is an IgM type antibody, and C in FIG. 3 is an IgA type antibody.

FIG. 4 is a graph showing the results of detection of a neutralizing autoantibody against human IL-33 at the recognition site of IL-33 protein, wherein A in FIG. 4 is the result of detection of P1 recognition, B in FIG. 4 is the result of detection of P2 recognition, and C in FIG. 4 is the result of detection of P3 recognition.

FIG. 5 is a graph showing the results of measurement of the neutralizing activity of IL-33 activity by an anti-human IL-33 neutralizing autoantibody, wherein A in FIG. 5 is the result of measurement of IL-5 and B in FIG. 5 is the result of measurement of IL-33.

FIG. 6 is a graph showing the results of measurements of the correlation between the concentration of cytokines in serum and IL-33 autoantibodies in patients with mild asthma, wherein A in FIG. 6 is the result of IL-4 measurement, B in FIG. 6 is the result of IL-9 measurement, C in FIG. 6 is the result of IL-13 measurement, D in FIG. 6 is the result of IL-25 measurement, and E in FIG. 6 is the result of IL-33 measurement.

FIG. 7 is a graph of the results of measurements of the IgE concentration in serum and the correlation of IL-33 autoantibodies in light asthma patients.

FIG. 8 shows the results of the experimental detection of IL-33-induced pneumonia in mice by neutralizing autoantibodies against human IL-33, wherein A in FIG. 8 is the lymphocyte count in lavage fluid of mouse lung, B in FIG. 8 is the detection result of IL-5 in serum, C in FIG. 8 is the detection result of IL-13 in serum, and D in FIG. 8 is the detection result of TSLP in serum.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. 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 application. Molecular biology and immunology techniques used in the examples, such as molecular cloning techniques like PCR, restriction enzyme digestion, ELISA, cell separation and culture, flow cytometry sorting, etc., are all methods commonly used in the art and are not described herein; materials, reagents and the like used in examples are commercially available unless otherwise specified.

The application provides an anti-human IL-33 neutralizing autoantibody, wherein a heavy chain variable region of the anti-human IL-33 neutralizing autoantibody comprises a heavy chain variable region shown as SEQ ID NO:1 and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

It is understood that an anti-human IL-33 neutralizing autoantibody comprises a heavy chain (H chain) comprising a heavy chain variable region (VH) and a light chain (L chain) comprising a light chain variable region (VL) wherein a site capable of specific binding to an antigen, i.e., an antigen binding site, is present to determine the specificity of antibody recognition.

The anti-human IL-33 neutralizing autoantibody provided by the application is discovered and obtained from B cells in blood of a patient by an inventor, is a natural human antibody, does not contain a heterologous component in an anti-human IL-33 neutralizing autoantibody sequence, does not cause rejection immune reaction of a human body in the using process, and is safer and more effective; the anti-human IL-33 neutralizing autoantibody provided by the application is a neutralizing antibody, and has wide application prospect in preparation of products for diagnosing, preventing or treating IL-33 related diseases.

In an embodiment of the present application, the gene encoding the heavy chain variable region of the anti-human IL-33 neutralizing autoantibody comprises the amino acid sequence set forth in SEQ ID NO: 5. It will be appreciated that the genes encoding the heavy chain variable region of the anti-human IL-33 neutralizing autoantibodies should take into account degenerate bases, i.e. the genes encoding the amino acid sequence shown in SEQ ID NO:1 include the nucleotide sequence shown in SEQ ID NO:5, and the scope of protection should also protect nucleotide sequences having base degeneracy with SEQ ID NO:5, the corresponding amino acid sequences of these nucleotide sequences remaining as SEQ ID NO: 1.

In an embodiment of the present application, the gene encoding the light chain variable region of the anti-human IL-33 neutralizing autoantibody comprises the amino acid sequence as set forth in SEQ ID NO: 6. It will be appreciated that the genes encoding the light chain variable region of the anti-human IL-33 neutralizing autoantibodies should take into account degenerate bases, i.e. the genes encoding the amino acid sequence shown in SEQ ID NO:2 include the nucleotide sequence shown in SEQ ID NO:6, and the scope of protection should also protect nucleotide sequences having base degeneracy with SEQ ID NO:6, the corresponding amino acid sequences of these nucleotide sequences remaining SEQ ID NO: 2.

In embodiments of the present application, the anti-human IL-33 neutralizing autoantibodies further comprise a heavy chain constant region. Further, the heavy chain constant region is hIgG1, hIgG2, hIgG3, or hIgG 4. The anti-human IL-33 neutralizing autoantibody is of IgG type, has good stability and long half life, and is beneficial to the application thereof. Further, the heavy chain constant region is hIgG 1.

In an embodiment of the present application, the heavy chain of an anti-human IL-33 neutralizing autoantibody comprises the amino acid sequence as set forth in SEQ ID NO: 3. Further, the gene encoding the heavy chain of the anti-human IL-33 neutralizing autoantibody includes the amino acid sequence shown in SEQ ID NO: 7. It will be appreciated that the genes encoding the heavy chain of an anti-human IL-33 neutralizing autoantibody should take into account degenerate bases, i.e. the gene encoding the amino acid sequence shown in SEQ ID NO. 3 comprises the nucleotide sequence shown in SEQ ID NO. 7, and the scope of protection should also protect nucleotide sequences having base degeneracy with SEQ ID NO. 7, the amino acid sequences corresponding to these nucleotide sequences still being SEQ ID NO. 3.

In embodiments of the present application, the anti-human IL-33 neutralizing autoantibodies further comprise a light chain constant region. In the present embodiment, the light chain of the anti-human IL-33 neutralizing autoantibody is a kappa chain or a lambda chain.

In an embodiment of the present application, the light chain of an anti-human IL-33 neutralizing autoantibody comprises the amino acid sequence set forth in SEQ ID NO: 4. Further, the gene encoding the light chain of the anti-human IL-33 neutralizing autoantibody includes the amino acid sequence shown in SEQ ID NO: 8. It will be appreciated that the genes encoding the light chain of the anti-human IL-33 neutralizing autoantibodies should take into account degenerate bases, i.e. the gene encoding the amino acid sequence shown in SEQ ID NO. 4 comprises the nucleotide sequence shown in SEQ ID NO. 8, and the scope of protection should also protect nucleotide sequences having base degeneracy with SEQ ID NO. 8, the amino acid sequences corresponding to these nucleotide sequences still being SEQ ID NO. 4.

In the present embodiment, the anti-human IL-33 neutralizing autoantibodies are naturally human antibodies. The inventor of the application generates the anti-human IL-33 neutralizing autoantibody through a genetic engineering method or through in vitro induction of human B cells, and the anti-human IL-33 neutralizing autoantibody is a natural human antibody, does not contain a heterologous fragment, does not cause the immune response of a human body, and is safer and more effective.

The application provides a preparation method of the anti-human IL-33 neutralizing autoantibody, which comprises obtaining the neutralizing autoantibody through genetic engineering or in vitro induction.

In embodiments of the present application, genetically engineered methods generate anti-human IL-33 neutralizing autoantibodies comprising: b cells for generating the anti-human IL-33 neutralizing autoantibody are obtained by identification, coding genes of heavy chain and light chain variable regions of the anti-human IL-33 neutralizing autoantibody are generated by a single cell PCR method, the coding gene of the heavy chain variable region of the anti-human IL-33 neutralizing autoantibody is inserted into an expression plasmid vector for coding a human antibody heavy chain constant region sequence, and the coding gene of the light chain variable region of the anti-human IL-33 neutralizing autoantibody is inserted into an expression plasmid vector for coding a human antibody light chain constant region sequence; the plasmid vector encoding the heavy chain and the plasmid vector encoding the light chain are transfected into a mammal host cell at the same time, and a complete anti-human IL-33 neutralizing autoantibody is obtained through protein expression and purification. Specifically, the heavy chain constant region is hIgG1, hIgG2, hIgG3 or hIgG4, the mammalian host cell may be, but is not limited to, HEK293, the expression plasmid vector encoding the human antibody heavy chain constant region sequence may be, but is not limited to, constructed according to the method of reference 6, or may be a commercially available expression plasmid such as AbVec-hIgG1(NTCC), pFUSE-CHIg (Invivogen), etc., the expression plasmid vector encoding the human antibody light chain constant region sequence may be, but is not limited to, constructed according to the method of reference 6, or may be a commercially available expression plasmid such as AbVec-hIgkappa (NTCC), pFUSE2-CLIg-hk (Invivogen), etc.

In an embodiment of the present application, a method for obtaining a neutralizing autoantibody against human IL-33 by in vitro induction, comprises: the IL-33 epitope fragment is used as immunogen to perform in vitro immunization on separated human peripheral blood mononuclear cells and induce to generate an anti-human IL-33 neutralizing autoantibody. Further, the amino acid sequence of the specific IL-33 epitope peptide fragment for inducing the generation of the anti-human IL-33 neutralizing autoantibody is at least one of ETTKRPSLKTGRKH, DLKKDEKKDKVLLS and NNKEHSVELHKCEK.

The present application provides a nucleic acid molecule encoding an antibody as described above. In embodiments of the present application, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region. In one embodiment, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:5 or a nucleotide sequence with base degeneracy with SEQ ID NO. 5. Further, the nucleic acid molecule comprises the nucleotide sequence as set forth in SEQ ID NO:7 or a nucleotide sequence with base degeneracy with SEQ ID NO. 7. In embodiments of the present application, the nucleic acid molecule comprises a nucleotide sequence encoding a light chain variable region. In one embodiment, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:6 or a nucleotide sequence with base degeneracy with SEQ ID NO. 6. Further, the nucleic acid molecule comprises the nucleotide sequence as set forth in SEQ ID NO:8 or a nucleotide sequence with base degeneracy with SEQ ID NO. 8.

The present application provides expression cassettes, recombinant vectors or recombinant cells comprising the above-described nucleic acid molecules. Anti-human IL-33 neutralizing autoantibodies are produced and produced by storing nucleic acid molecules encoding anti-human IL-33 neutralizing autoantibodies in expression cassettes, recombinant vectors or recombinant cells.

In embodiments of the present application, the expression cassette comprises a nucleic acid molecule as described above. Further, the expression cassette further comprises at least one of a promoter upstream of the nucleic acid molecule and a terminator downstream of the nucleic acid molecule.

In the present embodiments, the recombinant cell may be, but is not limited to, a recombinant eukaryotic cell.

The present application provides a pharmaceutical composition comprising an anti-human IL-33 neutralizing autoantibody as described above. By providing a pharmaceutical composition containing an anti-human IL-33-neutralizing autoantibody, the use thereof for the prevention or treatment of an IL-33 related disease is facilitated.

In the present embodiment, the pharmaceutical composition further comprises a pharmaceutical carrier for loading the anti-human IL-33 neutralizing autoantibody. Optionally, the carrier comprises at least one of a solvent, a polymer, and a liposome. Specifically, solvents include, but are not limited to, water, physiological saline, and other non-aqueous solvents; polymers may include, but are not limited to, polylysine, polyethyleneimine and modifications thereof, chitosan, polylactic acid, gelatin; liposomes may include, but are not limited to, cholesterol, soy lecithin, egg yolk lecithin. In one embodiment, the pharmaceutical carrier further comprises at least one of a diluent and an excipient. Further, the diluent includes at least one of starches, sugars, celluloses and inorganic salts; the excipient comprises binder, filler, lubricant in tablet, matrix part in semisolid ointment and cream, and at least one of antiseptic, antioxidant, correctant, aromatic, cosolvent, emulsifier and colorant in liquid preparation.

In the embodiments of the present application, the mass fraction of the anti-human IL-33 neutralizing autoantibodies in the pharmaceutical composition is 10% to 95%. Furthermore, the mass fraction of the anti-human IL-33 neutralizing autoantibody in the pharmaceutical composition is 20-80%, 25-75% or 30-60%.

In embodiments of the present application, the pharmaceutical composition may further comprise a second active ingredient. The second active ingredient is selected according to the use of the pharmaceutical composition and is not limited herein. Specifically, but not exclusively, when the pharmaceutical composition is used for allergic asthma, the second active ingredient has an effect of treating allergic asthma; when the pharmaceutical composition is used for allergic dermatitis, the second active ingredient has an effect of treating allergic dermatitis, and the like.

In an embodiment of the present application, the form of the pharmaceutical composition includes at least one of a tablet, a capsule, a powder, a granule, a pill, a syrup, a solution, a suspension, and an aerosol.

The application provides application of the anti-human IL-33 neutralizing autoantibody, the nucleic acid molecule, the expression cassette, the recombinant vector, the recombinant cell or the pharmaceutical composition in preparing a reagent for diagnosing or prognostically evaluating IL-33 mediated diseases. In one embodiment, the anti-human IL-33 neutralizing autoantibodies, nucleic acid molecules, expression cassettes, recombinant vectors or recombinant cells described above can be used to produce reagents that can be used to diagnose IL-33 mediated diseases or to prognosticate IL-33 mediated diseases.

The application provides an application of the anti-human IL-33 neutralizing autoantibody, the nucleic acid molecule, the expression cassette, the recombinant vector, the recombinant cell or the pharmaceutical composition in preparing a medicine for preventing or treating IL-33 mediated diseases. In one embodiment, the anti-human IL-33 neutralizing autoantibodies, nucleic acid molecules, expression cassettes, recombinant vectors, recombinant cells or pharmaceutical compositions described above can be used to prepare a medicament for preventing the development of IL-33 mediated diseases or for treating IL-33 mediated diseases.

In the embodiments of the present application, the IL-33 mediated diseases may include, but are not limited to, infectious diseases including septic shock, pneumonia caused by influenza virus, and the like, autoimmune diseases including systemic lupus erythematosus, colitis, scleroderma, rheumatoid arthritis, and the like.

Example 1 preparation of neutralizing autoantibodies against human IL-33 by genetic engineering

Three major IL-33 epitope peptides were designed and synthesized by epitope prediction software (DNAStar Protean software, SOPMA, and the Bepi Pred2.0), and the results are shown in Table 1. Enzyme-linked immunosorbent assay (ELISA) shows that the three IL-33 epitope peptides can be identified by the serum sample of the anti-IL-33 antibody positive patient, and the three IL-33 epitope peptides are proved to have high antigenicity and immunogenicity and can induce the generation of the IL-33 antibody. Therefore, the three IL-33 epitope peptides can be used as antigens to detect the level of anti-human IL-33 neutralizing autoantibodies in blood of patients, can also be used for activating and amplifying IL-33 specific B cells to generate anti-human IL-33 neutralizing autoantibodies, and can also be used for obtaining IL-33 specific B cells from blood through the sorting of flow cells after fluorescent labeling for cloning anti-human IL-33 neutralizing autoantibody genes.

TABLE 1 IL-33 epitope peptide information

Serial number	Name (R)	Length of amino acid sequence	Amino acid sequence
				P1	hIL-33 peptide start at 63aa	14	ETTKRPSLKTGRKH
P2	hIL-33 peptide start at 149aa	14	DLKKDEKKDKVLLS
				P3	hIL-33 peptide start at 197aa	14	NNKEHSVELHKCEK

PCR primers were designed based on the sequences of the light chain variable region and the heavy chain variable region of the antibody reported in reference 6. IL-33 autoantibodies producing Peripheral Blood Mononuclear Cells (PBMC) from asthmatic patients were collected. Since IL-33 autoantibodies of patients can recognize IL-33 epitope peptides in Table 1, IL-33 epitope peptides in Table 1 were fluorescently labeled and IL-33-specific CD19 of patients was found and obtained by flow-type single cell sorting⁺IgG⁺CD27⁺Memory B cells were amplified using a mature single B cell RT-PCR protocol (see reference 6 for details) using the QIAGEN OneStep RT-PCR kit to obtain anti-human IL-33 neutralizing autoantibody IgG heavy and light chain variable region cDNA sequences. The obtained IgG light chain variable region and heavy chain variable region fragments were detected by 1.5% agarose gel electrophoresis, and the light chain variable region and heavy chain variable region fragments were about 400 bp. The resulting DNA fragment was confirmed to be an antibody variable region by gel cutting, DNA sequencing and analysis (IgBLAST and http:// www.imgt.org/IMG T _ vquest/vquest).

The main IgG variable region cDNA fragments obtained above are subjected to a nested PCR reaction to amplify all IgG variable regions and introduce restriction enzyme sites by finding out naturally paired antibody light/heavy chain variable region genes. The antibody light/heavy chain variable region genes were inserted into expression vectors containing conserved regions of light/heavy chains of IgG1, respectively, via the introduced restriction enzyme sites. Such expression vectors typically contain eukaryotic gene expression promoters and enhancers, the site of insertion of the expressed gene, and the IgG1 light/heavy chain conserved region DNA sequences and termination codes. Wherein the heavy chain variable region of the produced anti-human IL-33 neutralizing autoantibody comprises the amino acid sequence as set forth in SEQ ID NO:1 and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO:2, or a pharmaceutically acceptable salt thereof. And then the constructed expression vector plasmid is cotransfected into eukaryotic cell HEK-293 cells to be expressed and generate fully human monoclonal IgG1 type anti-human IL-33 neutralizing autoantibodies.

Example 2 in vitro Induction of neutralizing autoantibodies to human IL-33

Human Peripheral Blood Mononuclear Cells (PBMC) are immunized with an in vitro antigen to induce the production of specific antibodies, a method for producing human specific antibodies [7], and we used and improved this in vitro immunization method to produce neutralizing autoantibodies against human IL-33. Briefly, PBMCs were isolated from healthy donors negative for neutralizing autoantibodies against human IL-33 and immunosuppressive cells were removed by Leu-Leu methyl ester hydrobromic acid (LLME). Then, three IL-33 epitope peptides (Table 1) were mixed with TLR9 ligand CpG-ODN (1. mu.M), cytokine IL-2(50ng/ml), and IL-4(50ng/ml), respectively, and after 7 days of in vitro immune cells, the supernatants were collected to obtain anti-human IL-33 neutralizing autoantibodies.

Effects of the embodiment

To evaluate the effect of the anti-human IL-33 neutralizing autoantibodies as claimed herein, the following effect examples were performed.

PBMCs were isolated from healthy donors negative for neutralizing autoantibodies against human IL-33 and immunosuppressive cells were removed with Leu-Leu methyl ester hydrobromic acid (LLME). Then using three IL-33 antigen peptides (Table 1) with different doses to act with TLR9 ligand CpG-ODN (1 μ M), cytokine IL-2(50ng/ml) and IL-4(50ng/ml) separately or together, collecting supernatant after 7 days in vitro immune cells, detecting the concentration of neutralizing autoantibodies against human IL-33 by ELISA, and the results are shown in FIG. 1, wherein the first group is peripheral blood mononuclear cells, the second group is peripheral blood mononuclear cells added with TLR9 ligand CpG-ODN, the third group is peripheral blood mononuclear cells added with cytokine IL-2, the fourth group is peripheral blood mononuclear cells added with cytokine IL-4, the fifth group is peripheral blood mononuclear cells added with TLR9 ligand CpG-ODN, cytokine IL-2, IL-4 and 0.1 μ g/ml epitope peptide, the sixth group is that the peripheral blood mononuclear cells are added with TLR9 ligand CpG-ODN, cytokine IL-2, IL-4 and 1 mu g/ml epitope peptide, the seventh group is that the peripheral blood mononuclear cells are added with TLR9 ligand CpG-ODN, cytokine IL-2, IL-4 and 5 mu g/ml epitope peptide, the eighth group is that the peripheral blood mononuclear cells are added with TLR9 ligand CpG-ODN, cytokine IL-2, IL-4 and 10 mu g/ml epitope peptide, wherein A in figure 1 is the detection result of adding human IL-33 polypeptide antigen 1(P1), in FIG. 1, B is the result of detection of antigen 2(P2) added with human IL-33 polypeptide, in fig. 1, C is the result of the detection of the added human IL-33 polypeptide antigen 3(P3), and the data represent 3 independent experiments, P <0.05, P <0.01 and PBMC or cytokine alone groups as controls. IL-2, IL-4, and TLR9 signaling may enhance B cell proliferation, differentiation to plasma cells, and switching of antibodies from IgM to IgG. As can be seen in figure 1, CpG or cytokine alone had no effect on IL-33 autoantibody production, nor did antigen alone have a significant effect on antibody induction (data not shown). However, the IL-33 epitope peptide antigen together with cytokines and CpG can induce the production of anti-IL-33 antibodies. The three epitope peptides of IL-33 can be recognized by anti-human IL-33 neutralizing autoantibodies of asthmatics, and can also induce anti-human IL-33 neutralizing autoantibodies. The result of the antigen dose-effect research shows that the optimal antigen doses of different IL-33 epitope peptides for inducing anti-human IL-33 neutralizing autoantibodies through immunity are different. This result indicates that IL-33 polypeptide antigens can induce human B cells to produce IgG type anti-human IL-33 neutralizing autoantibodies in vitro in the presence of CpG and cytokines IL-2, IL-4. This method can be used not only to produce anti-IL-33 antibody directly, but also to obtain IL-33 antibody-producing B cells, and then monoclonal antibody variable regions are cloned as in example 1, and monoclonal anti-human IL-33 neutralizing autoantibodies are produced by genetic engineering methods.

Autoantibody titers against 4 key Th2 cytokines in sera of 36 mild allergic asthma patients and 96 healthy controls were measured by the luciferase immunoprecipitation system (LIPS, detailed methods see reference 8) and expressed as Relative Luminescence Units (RLU) per microliter of serum, while IL-33 autoantibody titers in sera of 182 severe allergic asthma patients and 96 healthy controls were determined and compared to the percentage of anti-IL 33 positive samples of mild and severe allergic asthma patients, which were classified into mild allergic asthma patients and severe allergic asthma patients according to diagnostic criteria [9-10 ]. The assay results are shown in figure 2, where 95% of the serum sample titers of healthy controls were taken as the upper limit of normal (dashed line in figure 2), a-D in figure 2 are the assay results for autoantibodies in mild allergic asthma patients, a in figure 2 is the assay results for anti-IL-4 antibodies, B in figure 2 is the assay results for anti-IL-5 antibodies, C in figure 2 is the assay results for anti-IL-13 antibodies, D in figure 2 is the assay results for anti-IL-33 antibodies, E in figure 2 is the assay results for anti-IL-33 autoantibodies in severe allergic asthma patients, F in figure 2 is the positive rate for anti-IL-33 autoantibodies in mild and severe allergic asthma patients, data are mean ± Standard Deviation (SD), representing three independent experiments, P <0.05,/P <0.01,/P < 0.001. Among the 4 serum cytokine autoantibody assay results, only the anti-human IL-33 autoantibody titers in the mild asthma patient sera were significantly different from those of the healthy control group (D in fig. 2), and the serum titers of other inflammatory and regulatory cytokine autoantibodies were not significantly different. This indicates that allergic asthma patients selectively produce IL-33 autoantibodies. In addition, the differences between the mild-form allergic asthma patients and 182 severe-form allergic asthma patients were compared, and it was found that very few (6%) of the severe-form asthmatics produced IL-33 autoantibodies, significantly lower than the mild-form asthmatics (28%) (F in fig. 2). It can be seen that mainly mild allergic asthma patients produce neutralizing autoantibodies against human IL-33. Therefore, anti-human IL-33 neutralizing autoantibodies can be used as biomarkers of allergic asthma to distinguish patients with mild asthma from patients with severe asthma and to evaluate the prognosis of asthma patients with disease progression.

The results of ELISA for determination of neutralizing autoantibodies against human IL-33 in light and heavy allergic asthma patients, which were of type indicated in fig. 3, wherein a is an antibody of type IgG, B is an antibody of type IgM in fig. 3, C is an antibody of type IgA in fig. 3, data are mean ± SD, representing three independent experiments, < P <0.05, < P <0.01, < P <0.001, < P < 10 cases of IL-33 antibody positive sera of light and heavy asthmatic patients found in fig. 2, and then the antibody titer (OD) was determined after the addition of luciferase-labeled anti-human IgG, IgM or IgA antibodies and substrate. Antibody type analysis showed that patients with mild and severe asthma produced different levels of IL-33 autoantibodies, and that patients with severe asthma serum IgM autoantibodies produced significantly higher levels of IL-33 autoantibodies than patients with mild asthma, indicating that these antibodies were newly produced, most likely following development of severe asthma (B in figure 3). Thus, fig. 2 and 3 illustrate that IL-33 autoantibodies and antibody types can be used as biomarkers of allergic asthma to differentiate between mild and severe asthma, and prognosis of disease progression in asthmatic patients.

Three peptide fragments of the major epitope of IL-33 shown in table 1 were synthesized, P1, P2 and P3 peptide antigens were coated on an ELISA plate overnight, and IL-33 peptide antigen-specific assays were performed using ELISA on serum from 10 patients with mild asthma who were positive for neutralizing autoantibodies against human IL-33 and 3 sera from normal controls to determine the recognition sites of neutralizing autoantibodies against human IL-33 on IL-33 protein, as shown in fig. 4, where a in fig. 4 is the assay result for P1 recognition, B in fig. 4 is the assay result for P2 recognition, C in fig. 4 is the assay result for P3 recognition, each bar represents the results obtained from a single serum sample, data are mean ± SD representing three independent experiments,. P <0.05,. P <0.01,. P < 0.001. As can be seen from FIG. 4, all LIPS-detected asthma samples positive for IL-33 antibody were also positive by ELISA, and IL-33 antibody ELISA was also negative in the negative normal control group; all anti-IL-33 antibody positive serum samples were able to recognize the three major peptide antigens of IL-33, but the levels and patterns of epitopes recognized by different samples were different. The above data indicate that serum anti-human IL-33 neutralizing autoantibodies from asthmatic patients are polyclonal and recognize at least the three structural epitopes for IL-33 shown in Table 1.

To assess whether neutralizing autoantibodies against human IL-33 neutralizing autoantibodies in asthmatic patients, an antibody-mediated IL-33 in vitro neutralization assay was developed based on the ability of IL-33 to induce IL-5 production by activated T cells and to induce IL-33 production in macrophages via its receptor ST 2. In vitro cultured healthy human Peripheral Blood Mononuclear Cells (PBMCs) were used to test whether human serum anti-human IL-33 neutralizing autoantibodies block IL-33-induced IL-5 and IL-33 production. In human Peripheral Blood Mononuclear Cells (PBMC) activated by anti-CD 3 antibody with or without IL-33 recombinant protein stimulation, or anti-human IL-33 neutralizing autoantibodies and IL-33 antibody positive serum were cultured for 24 hours, cellular RNA was extracted, and the neutralizing ability of anti-human IL-33 neutralizing antibody and IL-33 antibody positive serum (n ═ 10) to IL-33 activity was examined by qPCR method, i.e., IL-33-induced IL-5 and IL-33mRNA expression levels were examined by qPCR, as shown in fig. 5, with data as mean ± SD, representing three independent experiments. P <0.05, P <0.01, P <0.001, wherein the first group is peripheral blood mononuclear cells, the second group is peripheral blood mononuclear cells added with IL-33 recombinant protein, the third group is peripheral blood mononuclear cells added with IL-33 recombinant protein and anti-human IL-33 neutralizing antibody, the fourth group is peripheral blood mononuclear cells added with IL-33 recombinant protein and anti-human IL-33 neutralizing autoantibody positive serum, the fifth group is peripheral blood mononuclear cells added with IL-33 recombinant protein and anti-human IL-33 neutralizing autoantibody negative serum, wherein a in fig. 5 is the detection result of IL-5, and B in fig. 5 is the detection result of IL-33. The results show that asthma serum samples positive for anti-human IL-33 neutralizing autoantibodies and anti-human IL-33 neutralizing autoantibodies significantly inhibited IL-33-induced mRNA expression of IL-5 and IL-33, but asthmatic patients had no inhibitory effect on serum negative for anti-human IL-33 neutralizing autoantibodies. The results of these 10 serum samples from patients with mild asthma collectively indicate that all anti-human IL-33 neutralizing autoantibodies raised by asthmatics have IL-33 activity neutralizing effects. Thus, anti-human IL-33 neutralizing autoantibodies are neutralizing antibodies.

The results of the detection of key cytokines in serum cytokine concentrations (9 of IL-33 autoantibodies positive and 26 of them negative) in 35 patients with mild asthma using Milliplex map cytokine test kit (8-plex) are shown in fig. 6, with data as mean ± SD,. P <0.05,. P <0.01, in fig. 6 a the detection of IL-4, in fig. 6B the detection of IL-9, in fig. 6C the detection of IL-13, in fig. 6D the detection of IL-25, and in fig. 6E the detection of IL-33. The concentrations of the asthmatic pathology-associated cytokines IL-4, IL-13, IL-25 and IL-33 in the serum of anti-human IL-33 neutralizing autoantibody positive patients were significantly reduced, but the change in IL-9 concentration was not significant, compared to anti-human IL-33 neutralizing autoantibody negative asthmatics. IgE plays an important role in the promotion of allergic asthma attack, and serum IgE levels of these 35 asthmatics were measured by ELISA, and the results are shown in fig. 7, with mean ± SD, P <0.05, and P < 0.01. The IgE concentration in serum of the anti-human IL-33 neutralizing autoantibody positive sample is obviously lower than that of the anti-human IL-33 neutralizing autoantibody negative asthma patient sample. These results are consistent with the in vitro function of neutralizing autoantibodies against human IL-33 and indicate that neutralizing autoantibodies against human IL-33 have a protective effect against asthma and not only reduce pathological type II cytokine and IgE levels, but also inhibit production of self-IL-33.

Human IL-33 can induce asthma-like changes such as mouse lung inflammation, IL-5 and IL-13 production by lung inflammatory cells [3], so that the method can be used for researching whether the neutralizing autoantibody of the human IL-33 can effectively neutralize the activity of IL-33 and relieving asthma pathological changes induced by the human IL-33. BALB/c mice at 6 weeks were randomly divided into 4 groups, the first being a saline control group; the second group is IL-33 recombinant protein (rIL-33) with the dosage of 0.4 mug/mouse; the third group was a rIL-33+ IgG (2. mu.g/mouse) control group; the fourth group was rIL-33+ anti-human IL-33 neutralizing autoantibodies (2. mu.g/mouse). By nasal administration, rIL-33+ anti-human IL-33 neutralizing autoantibody or control IgG was mixed and incubated for 30 minutes before nasal administration, and the experiment was completed after 3 days, wherein the detection results are shown in FIG. 8, in which A in FIG. 8 is lymphocyte count in mouse lung lavage fluid (BALF), B in FIG. 8 is detection result of IL-5 in serum, C in FIG. 8 is detection result of IL-13 in serum, and D in FIG. 8 is detection result of TSLP in serum. The IL-33-induced inflammatory lymphocyte infiltration of respiratory tract was significantly reduced by the anti-human IL-33 neutralizing autoantibodies (A in FIG. 8) compared to the control group, and the IL-33-induced inflammatory cytokines IL-5, IL-13 and TSLP levels in serum (B-D in FIG. 8) were effectively reduced by the anti-human IL-33 neutralizing autoantibodies. Therefore, the anti-human IL-33 neutralizing autoantibody has good IL-33 neutralizing effect in vivo, can effectively block IL-33 induced pneumonia and asthma-like pathological changes, and has preventing and treating effects on asthma.

Reference documents:

1.Holgate ST,Wenzel S,Postma DS,Weiss ST,Renz H,Sly PD.Asthma.Nat Rev Dis Primers.2015,1(1):15025.

2.Liew FY,Girard JP,Turnquist HR.Interleukin-33in health and disease.Nat Rev Immunol.2016.16(11):676-689.

3.Schmitz,J.,A.Owyang,E.Oldham,Y.Song,E.Murphy,T.K.McClanahan,G.Zurawski,M.Moshrefi,J.Qin,X.Li,et al IL-33,an interleukin-1-like cytokine that signals via the IL-1receptor-related protein ST2 and induces T helper type 2-associated cytokines.Immunity 2005.23:479-490.

4.Kurowska-Stolarska M,Kewin P,Murphy G,Russo RC,Stolarski B,Garcia CC,Komai-Koma M,Pitman N,Li Y,Niedbala W,McKenzie AN,Teixeira MM,Liew FY,Xu D.IL-33induces antigen-specific IL-5+T cells and promotes allergic-induced airway inflammation independent of IL-4.J Immunol.2008.181(7):4780-90

5.Li D,Guabiraba R,Besnard AG,Komai-Koma M,Jabir MS,Zhang L,Graham GJ,Kurowska-Stolarska M,Liew FY,McSharry C,Xu D.IL-33 promotes ST2-dependent lung fibrosis by the induction of alternatively activated macrophages and innate lymphoid cells in mice.J Allergy Clin Immunol.2014.134(6):1422-1432.e11.

6.Tiller T,Meffre E,Yurasov S,Tsuiji M,Nussenzweig MC,Wardemann H.Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.J Immunol Methods.2008,329(1-2):112-124.

7.Tomimatsu K,Shirahata S.Antigen-specific in vitro immunization:a source for human monoclonal antibodies.Methods Mol Biol.2014.1060:297-307.

8.Burbelo PD,Goldman R,Mattson TL。A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins.BMC Biotechnol.2005.5:22.

9.Kuruvilla ME,Lee FE,Lee GB.Understanding Asthma Phenotypes,Endotypes,and Mechanisms of Disease.Clin Rev Allergy Immunol.2019.56(2):219-233.

10.Chung KF.Diagnosis and Management of Severe Asthma.Semin Respir Crit Care Med.2018.39(1):91-99.

while the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

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Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

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Lys Gly Pro Ser Val Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

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Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

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Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

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gcccctggac aagggcttga gtggatggga tggatcagcg cttacaatgg taacacaaag 180

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gtgaccgtgc cctccagcag cttgggcacc cagacctaca tctgcaacgt gaatcacaag 660

cccagcaaca ccaaggtgga caagagagtt gagcccaaat cttgtgacaa aactcacaca 720

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aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 960

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 1020

aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1080

ccacaggtgt acaccctgcc cccatcccgg gaggagatga ccaagaacca ggtcagcctg 1140

acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 1200

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1260

ctctatagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1320

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtccccg 1380

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tggtaccaac aaaaagcagg acagcctcct aagttgctca tttactggac atcaacccgg 180

gaatccgggg tccctgaccg attcagtggc agcgggcctg ggacaaactt cactctcacc 240

atcagcagcc tacaggctga agatgtggca gtttattatt gtcagcaata ttttagtagt 300

cccctgacgt tcggccaagg gaccaaggtg gaagtcaaac gaactgtggc tgcaccatct 360

gtcttcacgg tggctgcacc atctgtcttc atcttcccgc catctgatga gcagttgaaa 420

tctggaactg cctctgttgt gtgcctgctg aataacttct atcccagaga ggccaaagta 480

cagtggaagg tggataacgc cctccaatcg ggtaactccc aggagagtgt cacagagcag 540

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Claims

1. An anti-human IL-33 neutralizing autoantibody characterized in that the heavy chain variable region of said anti-human IL-33 neutralizing autoantibody is SEQ ID NO:1, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

2. The anti-human IL-33 neutralizing autoantibody of claim 1 further comprising a heavy chain constant region that is hIgG1, hIgG2, hIgG3, or hIgG 4.

3. The anti-human IL-33 neutralizing autoantibody of claim 2 wherein the heavy chain of said anti-human IL-33 neutralizing autoantibody is SEQ ID NO: 3.

4. The anti-human IL-33 neutralizing autoantibody of claim 1 wherein the light chain of said anti-human IL-33 neutralizing autoantibody is SEQ ID NO: 4.

5. A method for producing the neutralizing autoantibody against human IL-33 according to any one of claims 1 to 4, which comprises obtaining the neutralizing autoantibody by genetic engineering.

6. A nucleic acid molecule encoding an anti-human IL-33 neutralizing autoantibody according to any one of claims 1-4.

7. An expression cassette, recombinant vector or recombinant cell comprising the nucleic acid molecule of claim 6.

8. A pharmaceutical composition comprising an anti-human IL-33 neutralizing autoantibody according to any one of claims 1 to 4.

9. Use of the anti-human IL-33 neutralizing autoantibody according to any one of claims 1-4, the anti-human IL-33 neutralizing autoantibody produced by the production process according to claim 5, the nucleic acid molecule according to claim 6, the expression cassette, the recombinant vector or the recombinant cell according to claim 7, or the pharmaceutical composition according to claim 8 for the preparation of a reagent for the diagnosis or prognosis of an IL-33 mediated disease, wherein the IL-33 mediated disease is an infectious disease or an autoimmune disease.

10. The use of claim 9, wherein the infectious disease is septic shock or pneumonia caused by influenza virus;

the autoimmune disease is asthma, systemic lupus erythematosus, colitis, scleroderma or rheumatoid arthritis.

11. Use of the anti-human IL-33 neutralizing autoantibody according to any one of claims 1-4, the anti-human IL-33 neutralizing autoantibody produced by the production process according to claim 5, the nucleic acid molecule according to claim 6, the expression cassette, the recombinant vector or the recombinant cell according to claim 7, or the pharmaceutical composition according to claim 8 for the production of a medicament for the prophylaxis or treatment of an IL-33 mediated disease, wherein the IL-33 mediated disease is an infectious disease or an autoimmune disease.

12. The use of claim 11, wherein the infectious disease is septic shock or pneumonia caused by influenza virus;