CN114395042A

CN114395042A - anti-IL-33 humanized antibodies and uses thereof

Info

Publication number: CN114395042A
Application number: CN202210308391.8A
Authority: CN
Inventors: 王双; 王荣娟; 焦莎莎; 张畅; 张锦超
Original assignee: Beijing Konuoxincheng Technology Co ltd
Current assignee: Beijing Konuoxincheng Technology Co ltd
Priority date: 2022-03-28
Filing date: 2022-03-28
Publication date: 2022-04-26
Anticipated expiration: 2042-03-28
Also published as: CN114395042B

Abstract

The invention discloses an anti-human IL-33 antibody or antigen binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises antigenic determinant regions VH CDR1, VH CDR2 and VH CDR3, and the VL comprises antigenic determinant regions VL CDR1, VL CDR2 and VL CDR 3. The humanized anti-IL-33 antibody is derived from hybridoma, can bind to human IL-33 with high affinity, blocks the combination of IL-33 and ST2, inhibits the promotion effect of IL-33 on the expression of KU812 cell IL5, and provides candidate antibody molecules for treating IL-33 related diseases.

Description

anti-IL-33 humanized antibodies and uses thereof

Technical Field

The present invention relates to the field of biopharmaceuticals. In particular, the invention relates to novel artificially designed antibodies, in particular IL-33 humanized antibodies or antigen binding fragments. The invention also relates to the therapeutic and diagnostic use of these antibodies that bind to IL-33, in particular in the treatment, prevention and/or diagnosis of IL-33 related diseases, such as inflammation.

Background

Inflammation is a defensive response of living tissue with a vascular system to an injury factor. It is commonly known as "inflammation" which is a defense response of the body to irritation, manifested as redness, swelling, heat, pain. Vascular responses are a central link in the inflammatory process. Normally, inflammation is beneficial and an automatic defense response of the human body, but sometimes, inflammation is also harmful, such as attack on the body's own tissues, inflammation occurring in transparent tissues, and the like.

Interleukin-33 (IL-33) is a multifunctional gene discovered in 2005, is one of the important regulators of inflammatory response and immune bias, can function as a molecule located in the nucleus, functions as a transcription factor, and can be secreted to the outside of the cell to function as a cytokine. IL-33 is a novel member of the IL-1 family, and the receptor is ST2 (Interleukin 1 like receptor), and the research suggests that IL-33 is released after epithelial cells are damaged, and has multiple functions, including stimulating the recombination of epithelial cells and promoting the generation of inflammation. The IL-33/ST2 signaling pathway is a potential target for the treatment of asthma, atopic dermatitis, COPD and chronic obstructive pulmonary disease, chronic sinusitis, and rheumatoid arthritis. There are currently several projects directed to IL-33 and ST2 in clinical research. Including etokamicab by Anaptybio, SAR-440340 by Xenoffy, MEDI-3506 by MedImmune, and the like.

As the mechanism of action of IL-33 on the development of inflammatory diseases becomes clearer, therapeutic anti-human IL-33 antibodies attract more and more attention. At present, the clinical test is the fastest-progressing fully-humanized anti-IL-33 monoclonal antibody SAR-440340 developed by Xenoffy and regenerators, however, the clinical application prospect of the SAR-440340 in asthma, atopic dermatitis, chronic obstructive pneumonia and the like is still unclear, and the SAR-440340 is used together with dabbituo for treating asthma before, but the phase II clinical test has poor effect. Thus, anti-IL-33 antibodies are to be further investigated.

Disclosure of Invention

The present invention is intended to solve the problem of the prior art that IL-33 antibodies have unstable affinity for the human IL-33 receptor, and specifically, the problem of the binding activity of IL-33 antibodies. To this end, it is an object of the present invention to produce IL-33 antibodies or antigen-binding fragments with higher affinity.

Thus, according to one aspect of embodiments of the present invention there is provided an anti-human IL-33 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the antigenic determinant regions VH CDR1, VH CDR2 and VH CDR3, the VH CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 14, and the VH CDR2 comprises an amino acid sequence as set forth in EIFPGTGTTYYNX₁KFX₂G, and the VH CDR3 comprises an amino acid sequence shown as SEQ ID NO: 16; the VL comprises the antigenic determinants VL CDR1, VL CDR2 and VL CDR3, the VL CDR1 comprises as X₃ASSSVSYMH, and the VL CDR2 comprises an amino acid sequence as RTSNLX₄S, and the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13; wherein, X1 is E or Q; x2 is K or Q; x3 is S or R; x4 position A or Q.

According to the IL-33 antibody or antigen-binding fragment of the embodiment, the antibody obtained by the invention can be combined with human IL-33, and the combined activity of the antibody and the human IL-33 is better than that of a control antibody. The activity of a chimeric antibody ch25D8 of the 25D8 molecule for inhibiting the combination of ST2 and IL-33 is obviously superior to that of a control antibody, and meanwhile, the humanized antibody of the chimeric antibody also shows that the activity of inhibiting the IL-33 is superior to that of the control antibody in the detection of in vitro cytological activity, and has strong affinity.

In addition, the antibody or antigen-binding fragment according to the above embodiment of the present invention may have the following additional technical features:

according to an embodiment of the invention, said VH comprises the amino acid sequence as set forth in SEQ ID NO: 4, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 3.

According to an embodiment of the invention, said VH comprises the amino acid sequence as set forth in SEQ ID NO: 8, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 7. Further stated, the VH and VL sequences of the antibody or antigen-binding fragment can be as set forth in SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 7 or SEQ ID NO: 8, and (b) is obtained by performing insertion, deletion, mutation or modification of one or more amino acids based on the amino acid sequence shown in 8.

According to an embodiment of the invention, the antibody or antigen binding fragment thereof is a monoclonal antibody. Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone that are directed against only a particular epitope. For example, the antibodies of the present embodiments specifically bind only IL-33.

According to an embodiment of the invention, the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody. The humanized antibody is mainly a mouse monoclonal antibody which is modified by gene cloning and DNA recombination technology and combined with a human target after modification, and the re-expressed antibody has most of amino acid sequences replaced by human sequences, basically keeps the affinity and specificity of a parent mouse monoclonal antibody, reduces the heterogeneity thereof, and is favorable for being applied to a human body, for example, the antibody or antigen binding fragment of the embodiment of the invention can be combined with the specificity of human IL-33 after being humanized.

According to an embodiment of the invention, the antibody or antigen-binding fragment thereof is an antibody fragment that specifically binds human IL-33 and is selected from the group consisting of Fv, Fab ', scFv, and F (ab') 2.

According to another aspect of the invention, the invention provides an isolated polynucleotide. According to an embodiment of the invention, the isolated polynucleotide encodes an anti-human IL-33 antibody or antigen-binding fragment thereof as described above. According to an embodiment of the invention, the isolated polynucleotide encodes an IL-33 antibody or antigen-binding fragment as described in the previous paragraph. Thus, the polypeptide encoded by the polynucleotide can specifically bind to IL-33.

According to another aspect of the invention, the invention provides an isolated polynucleotide. According to embodiments of the invention, the isolated polynucleotide encodes a light chain variable region or a heavy chain variable region, or a light chain or a heavy chain, of an anti-human IL-33 antibody or antigen-binding fragment thereof.

According to another aspect of the present invention, there is provided a vector. According to an embodiment of the invention, the vector comprises one or more of the polynucleotides described above.

According to an embodiment of the invention, the vector comprises both of the foregoing polynucleotides and encodes a VL region and a VH region that together bind IL-33.

According to another aspect of the present invention, there is provided a pair of vectors. According to an embodiment of the invention, each vector pair comprises one of the foregoing polynucleotides, wherein the vector pairs together encode a VL region and a VH region that together bind IL-33.

According to another aspect of the present invention, there is provided a host cell. According to an embodiment of the invention, the host cell comprises the aforementioned vector, or the aforementioned pair of vectors.

According to another aspect of the present invention, there is provided a composition. According to an embodiment of the present invention, the composition comprises the anti-human IL-33 antibody or antigen-binding fragment thereof, the polynucleotide, the vector pair, or the host cell; and optionally pharmaceutically acceptable excipients. Therefore, the composition can be specifically combined with IL-33, and has good effect of inhibiting inflammatory factors and good drug effect.

According to another aspect of the present invention, the present invention provides a method for preparing an anti-human IL-33 antibody or a fragment thereof. According to an embodiment of the invention, the method comprises: 1) culturing the aforementioned host cell under suitable conditions; 2) separating and recovering the anti-human IL-33 antibody or the antigen-binding fragment thereof. The invention adopts a method for separating and purifying target protein after in vitro cell culture to obtain the antibody specifically combined with IL-33.

According to another aspect of the invention, the invention provides a use. According to an embodiment of the invention, the use comprises: the anti-human IL-33 antibody or a fragment thereof, the polynucleotide, the vector, the pair of vectors, the host cell and the composition are used for preparing a diagnostic reagent for inflammation or cardiovascular diseases.

According to another aspect of the invention, the invention provides a use. According to an embodiment of the invention, the use comprises: use of the anti-human IL-33 antibody or fragment thereof, the polynucleotide, the vector, the pair of vectors, the host cell and the composition in the preparation of a medicament for treating inflammation or cardiovascular disease.

According to an embodiment of the invention, the inflammation comprises asthma, atopic dermatitis, COPD and chronic obstructive pulmonary disease, chronic sinusitis and rheumatoid arthritis.

The present embodiments protect a humanized anti-IL-33 antibody from a hybridoma. The antibody is combined with human IL-33 with high affinity, blocks the combination of IL-33 and ST2, inhibits the promotion effect of IL-33 on the expression of IL5 of KU812 cells, overcomes the defects of insufficient affinity, specificity and adverse reaction of anti-human IL-33 antibodies in the prior art, has high stability, strong affinity and higher biological activity, and provides candidate antibody molecules for the treatment of IL-33 related diseases.

For a better understanding of the present invention, certain terms are first defined. Other definitions are listed throughout the detailed description section.

The term "specific" refers to the determination of the presence or absence of a protein in a heterogeneous population of proteins and/or other organisms. Thus, under the conditions specified, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

The term "antibody" herein is intended to include full-length antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof. Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH 3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain CL. The VH and VL regions can also be divided into hypervariable regions, called Complementarity Determining Regions (CDRs), which are separated by more conserved Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "antigen-binding fragment" of an antibody (or simply antibody portion), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind antigen. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments comprised in the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH 1; (ii) a F (ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a hinge region disulfide bridge; (iii) an Fd fragment consisting of VH and CH 1; (iv) an Fv fragment consisting of VL and VH antibody single arms; (v) dAb fragments consisting of VH (Ward et al, (1989) Nature 341: 544-546); (vi) an isolated Complementarity Determining Region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be joined by recombinant methods via a synthetic linker that makes the two single protein chains, in which the VL and VH regions pair to form monovalent molecules (known as single chain Fc (scFv); see, e.g., Bird et al., (1988) Science 242: 423-. These single chain antibodies are also intended to be included within the term meaning. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

Antigen-binding fragments of the invention include those capable of specifically binding to an antigen. Examples of antibody binding fragments include, for example, but are not limited to, Fab ', F (ab')₂Fv fragments, single chain Fv (scFv) fragments and single domain fragments.

The Fab fragment contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab 'fragments are generated by cleavage of the disulfide bond at the hinge cysteine of the F (ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art. Fab and F (ab')2 fragments lack the fragment crystallizable (Fc) region of intact antibodies, clear more rapidly from the circulation of animals, and may have less non-specific tissue binding than intact antibodies (see, e.g., Wahl et al, 1983, J. Nucl. Med. 24: 316).

As is generally understood in the art, an "Fc" region is a fragment crystallizable constant region of an antibody that does not comprise an antigen-specific binding region. In IgG, IgA and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of an antibody (CH 2 and CH3 domains, respectively). The IgM and IgE Fc regions contain three heavy chain constant domains (CH 2, CH3, and CH4 domains) in each polypeptide chain.

The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or fragment thereof, and an antigen). The term "binding affinity" is used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules (e.g., an antibody or fragment thereof, and an antigen) via a monovalent interaction can be quantitatively determined by determining the dissociation constant (KD). KD can then be determined by measurement of complex formation and dissociation kinetics, for example by SPR methods. The rate constants corresponding to the association and dissociation of monovalent complexes are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. KD is linked to ka and KD by the equation KD = KD/ka. According to the above definitions, the binding affinities associated with different molecular interactions, e.g. the binding affinities of different antibodies for a given antigen, can be compared by comparing the KD values of the individual antibody/antigen complexes. Similarly, the specificity of an interaction can be evaluated by determining and comparing the KD value for the interaction of interest (e.g., a specific interaction between an antibody and an antigen) to the KD value for an interaction not of interest. The value of the dissociation constant can be determined directly by well-known methods, e.g., by standard assays that assess the binding ability of a ligand (e.g., an antibody) to a target are known in the art and include, e.g., ELISA, western blot, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of the antibody can also be assessed by standard assays known in the art, such as SPR. A competitive binding assay may be performed in which the binding of an antibody to a target is compared to the binding of another ligand (e.g., another antibody) to the target.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:

FIG. 1 shows an IL-33/ST2 recombinant protein binding blocking assay according to one embodiment of the invention;

FIG. 2 shows that an anti-IL-33 antibody inhibits IL-33 production-promoting activity of KU812-IL5, according to one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

Anti-human IL-33 antibodies and antigen-binding fragments thereof

The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to IL-33. The anti-human IL-33 antibody or the antigen-binding fragment thereof according to the embodiment of the invention can be specifically combined with human IL-33 or mouse IL-33 protein, reduce the level of proinflammatory cytokines in serum of patients, reduce the growth of eosinophils and reduce inflammatory response.

The present embodiments provide anti-IL-33 antibodies, including the mouse antibody 25D8, as well as humanized and chimeric antibodies thereto. These chimeric antibodies have VH and VL from mouse antibodies. However, the constant domains of these chimeric antibodies are from human antibodies (e.g., human IgG1, human IgG2, human IgG3, or human IgG 4), and the chimeric antibody is labeled ch25D 8. In addition, the present examples also provide the humanized antibody H25D8H1L 1.

The CDR sequences (Kabat definition), heavy and light chain variable regions, and heavy and light chain constant regions of some of the disclosed antibodies are shown in table 1 below.

TABLE 1

Ab	VH-CDR1	VH-CDR2	VH-CDR3	VL-CDR1	VL-CDR2	VL-CDR3	VH	VL	CH	CL
											25D8	14	15	16	11	12	13	4	3	-	-
ch25D8	14	15	16	11	12	13	4	3	10	9
											h25D8H1L1	14	23	16	21	22	13	8	7	10	9

The embodiments of the present invention also provide the amino acid sequences of the heavy chain variable region and the light chain variable region of the humanized antibody. Because there are different ways to humanize a mouse antibody (e.g., the sequence can be modified with different amino acid substitutions), the heavy and light chains of an antibody can have more than one form of humanized sequence. The amino acid sequence of the heavy chain variable region of humanized antibody H25D8H1L1 is shown in SEQ ID NO: 8. The amino acid sequence of the light chain variable region of humanized antibody H25D8H1L1 is represented by SEQ ID NO: 7.

In some embodiments, an antibody may have a heavy chain variable region (VH) comprising or consisting of complementarity determining regions CDR1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence; the VL comprises or consists of a CDR1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VL CDR3 amino acid sequence.

"percent (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence shown in the specification, after aligning the candidate sequence with the specific amino acid sequence shown in the specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, or 99% or more identity, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variant may comprise conservative changes.

In some embodiments, the antibodies or antigen binding fragments described herein may comprise heavy and light chain variable regions comprising one, two or three CDRs selected from the group consisting of: SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 21. SEQ ID NO: 22 or SEQ ID NO: 23.

the embodiments also provide an antibody or antigen-binding fragment thereof that binds IL-33, wherein the antibody or antigen-binding fragment has VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR 3. In some embodiments, the sequences of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are determined based on various CDR definitions known in the art, e.g., Kabat definitions, Chothia definitions, or IMGT definitions. The sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are shown in table 1.

Nucleic acids, vectors and cells

The embodiments also provide nucleic acids comprising polynucleotides encoding polypeptides comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in table 1, or has sequences as shown in table 1. When a polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to IL-33 (e.g., human IL-33).

The embodiments also provide vectors comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors are introduced (i.e., such that the host cells contain the polynucleotides and/or polynucleotide-containing vectors), and production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into said host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, a polynucleotide of interest is targeted for expression in the vector by being operably linked to regulatory elements such as a promoter, enhancer and/or polyadenylation tail, which are located at or near or on both sides of the site of integration of the polynucleotide of interest within the vector or in the genome of the host cell, such that the polynucleotide of interest will be translated in the host cell into which the expression vector is introduced.

The vector may be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and/or transduction (e.g., with a recombinant virus). Thus, non-limiting examples of vectors include viral vectors (useful for the production of recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

The embodiments of the present invention provide host cells transformed with the vectors described above. The host cell may be a prokaryotic or eukaryotic cell. A preferred prokaryotic host cell is E.coli (Escherichia coli). Preferably, the eukaryotic cell is selected from: protist cells, animal cells, plant cells and fungal cells. More preferably, the host cell is a mammalian cell, including but not limited to CHO and COS cells. A preferred fungal cell is Saccharomyces cerevisiae.

Method for producing anti-IL-33 antibody

Isolated fragments of human IL-33 can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein may be conjugated to an agent that is immunogenic in the species to be immunized. The animal may be injected multiple times with the antigenic peptide or protein.

Immunogens are typically used to produce antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations may contain, for example, recombinantly expressed polypeptides or chemically synthesized polypeptides (e.g., fragments of human IL-33). The formulation may further comprise an adjuvant, such as freund's complete or incomplete adjuvant, or similar immunostimulant.

Pharmaceutical compositions and uses and methods of treatment

The embodiments also provide pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described in the embodiments. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf-life or effectiveness of the antibody.

In one aspect, the invention provides the use of an anti-human IL-33 antibody or antigen-binding fragment thereof as described above in the manufacture of a medicament for the treatment of inflammation or cardiovascular disease. According to an embodiment of the invention, the inflammation comprises asthma, atopic dermatitis, COPD and chronic obstructive pulmonary disease, chronic sinusitis and rheumatoid arthritis.

The present invention provides that one or more antibodies or antigen-binding fragments thereof can be used for a variety of therapeutic purposes. In one aspect, the present disclosure provides methods for treating inflammation in a subject, methods of reducing the risk of exacerbation of inflammation. In some embodiments, the treatment can stop, slow, or inhibit the progression of inflammation. In some embodiments, the treatment may result in a reduction in the number, severity, and/or duration of one or more symptoms of inflammation in the subject.

In one aspect, the disclosure features methods that include administering to a subject in need thereof (e.g., a subject having, or identified as having, or diagnosed with, inflammation such as asthma, atopic dermatitis, COPD, and chronic obstructive pulmonary disease, chronic sinusitis, and rheumatoid arthritis), a therapeutically effective dose of an antibody, or antigen-binding fragment thereof, disclosed herein.

In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of inflammation. A patient suffering from inflammation can be identified by a variety of methods known in the art.

Reagent kit

The embodiments also provide kits containing at least one (e.g., one, two, three, or four) of the IL-33 antibodies or antigen-binding fragments described in the embodiments. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in the kit in any combination. Kits may be prepared in any manner known in the art.

The cartridge may further comprise: (1) solid phase carriers (immunoadsorbents) coated with antigen or antibody; (2) an enzyme-labeled antigen or antibody (conjugate); (3) a substrate for an enzyme; (4) negative and positive controls (in the qualitative determination), reference standards and control sera (in the quantitative determination); (5) dilutions of the conjugate and the sample; (6) washing solution, in plate ELISA, the common diluent is phosphate buffered saline containing 0.05% tween 20; (7) the enzyme reaction stopping solution is sulfuric acid which is commonly used as HRP reaction stopping solution.

The invention provides application of the anti-human IL-33 antibody or antigen binding fragment kit thereof in a detection reagent. According to the embodiment of the invention, the detection comprises RT-PCR, Northern blot, Dot blot, Real time RT PCR, chip analysis, polyA screening, in-vitro translation, RNase protection analysis, molecular cloning and other detection experiments.

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.

Example 1 preparation of anti-IL-33 hybridoma murine chimeric antibodies

Preparation of control antibodies

According to the APE4909 sequence disclosed in the patent WO2015106080A2, the antibody sequence of a control antibody ANB020 is synthesized, and the sequences of the light chain variable region and the heavy chain variable region are shown as SEQ ID NO.1 and SEQ ID NO. 2. Respectively cloning the light and heavy chain sequences into a eukaryotic transient expression vector containing a human IgG1 light and heavy chain constant region to obtain control antibody light chain and heavy chain expression plasmids, transferring the control antibody light chain and heavy chain expression plasmids into escherichia coli for amplification, separating to obtain a large number of plasmids containing the control antibody light chain and heavy chain, utilizing the plasmids, and respectively transferring the light and heavy chain plasmids of the control antibody into HEK293 cells for recombinant expression according to the operation instruction of a transfection reagent 293fectin (Cat: 12347019, Gibco). 5-6 days after cell transfection, culture supernatant is taken and purified by a ProA affinity chromatography column to obtain a control antibody ANB 020.

SEQ ID NO.1 ANB020-VL

DIQLTQSPSFLSASVGDRVTLTCKASQDVGTAVAWTQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQAKTYPFTFGSGTKLEIK

SEQ ID NO.2 ANB020-VH

QVQLMQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGTIYPRNSNTDYNQKFKARVTMTRDTSTSTVYMELSSLRSEDTAVYYCARPLYYYLTSPPTLFWGQGTLVTVSS

Preparation of murine hybridoma antibodies

Balb/c mice were immunized with human IL-33 recombinant protein (SEQ ID NO: NP-001300973.1 aa112-aa 270), and cell fusion and hybridoma preparation were performed. Analyzing the hybridoma culture supernatant by ELISA, selecting specific positive clones to obtain specific positive clones 25D8 and 25H12, and extracting total RNA of hybridoma cells by a TRIzol kit (Cat: 15596026, Invitrogen); reverse transcribing hybridoma cell total RNA to cDNA using M-MuLV reverse transcriptase (Cat: M0253S, NEB); amplification of antibody light chain variable region IgVL (kappa) and heavy chain variable region V using degenerate primers and Phusion kit (Cat: E0553L, NEB)_HA sequence; purifying PCR amplification products by using a gel recovery kit (Cat: AP-GX-250, Axygen); connecting the amplified PCR product to T vector according to the specification of T vector cloning kit (Cat: ZC205, Chuanglian organism), transforming competent cells of Escherichia coli, amplifying strain, extracting plasmid, and performing DNA sequencing to obtain monoclonal antibodyA body variable region sequence.

The sequencing result shows that the amino acid sequence of the light chain variable region of the 25D8 murine antibody is shown in SEQ ID NO.3, and the sequence of the heavy chain variable region is shown in SEQ ID NO. 4. The amino acid sequence of the light chain variable region of the 25H12 murine antibody is shown in SEQ ID NO.5, and the sequence of the heavy chain variable region is shown in SEQ ID NO. 6.

SEQ ID NO.3 25D8VL

Qivltqspaimsaspgekvtitcsasssvsymhwfqqkpgtspklwiyrtsnlasgvpdrfsgsgsgtsysltisrmeaedaatyyclqrstylfmfgagtklelk

SEQ ID NO.4 25D8VH

qvqlqqsgtelvkpgasvklscktsgytftsywiqwvkqrpgqglgwigeifpgtgttyynekfkgkatlttdtssttaymqlssltsedsavyfcarrirdyygsnyfdywgqgttltvss

SEQ ID NO.5 25H12VL

qivltqspaimsaspgekvtitcsasssvsymhwfqqkpgtspklwiyrtsnlasgvpdrfsgsgsgtsysltisrmeaedaatyycqqrssylfmfgagtklelk

SEQ ID NO.6 25H12VH

evqlqqsgaelvkpgasvklsctasgfnikdtyihwvkqrpeqglewigridptadntkyhpkfqgkatltadtssntaylqlssltsedtavyfcargssyaldywgqgtsvtvss

Chimeric antibody identification

The light chain variable region and heavy chain variable region genes are introduced into enzyme cutting sites through PCR, and are respectively cloned into eukaryotic transient expression vectors containing the coding genes at the upstream of the human-kappa light chain constant region (SEQ ID NO. 9) and the human IgG1 heavy chain constant region (SEQ ID NO. 10) to obtain expression plasmids of human-mouse chimeric light chain (pKN 019-25D8L, pKN019-25H 12L) and human-mouse chimeric heavy chain (pKN 032-25D8H, pKN032-25H 12H), and the light chain plasmids and the heavy chain plasmids of the chimeric antibody are respectively transferred into HEK293 cells for recombinant expression according to the operation instructions of a transfection reagent 293fectin (Cat: 12347019, Gibco). 5-6 days after cell transfection, culture supernatant was taken and purified by a ProA affinity chromatography column to obtain ch25D8 and ch25H12 protein samples.

The affinity of the chimeric antibodies ch25D8 and ch25H12 was determined using fortebio. Specifically, the antibody affinity is determined by a method of capturing the Fc region of an antibody with a capture Antibody (AHC) bioprobe against the Fc region of an anti-human antibody. During measurement, the antibody was diluted to 4. mu.g/mL with PBS buffer and flowed over the surface of an AHC probe (Cat: 18-0015, PALL) for 120 s. Human IL33-his recombinant protein (SEQ ID NO: NP-001300973.1 aa112-aa 270) was used as a mobile phase, and IL33-his concentrations were 15, 30, 45, and 60nM, respectively. The binding time was 100s and the dissociation time was 300 s. After the experiment, blank control response values were deducted, and the software was run for 1: 1 Langmuir binding pattern was fitted and the kinetic constants for antigen-antibody binding are shown in Table 2 below.

SEQ ID NO.9 human-kappa light chain constant region

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO.10 human IgG1 heavy chain constant region

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

TABLE 2 chimeric antibody affinity assay

Sample ID	KD (M)	kon(1/Ms)	kdis(1/s)
				ch25D8	9.00E-10	8.36E+04	7.53E-05
ch25H12	7.77E-08	7.97E+03	6.19E-04
				ANB020	6.70E-09	6.25E+04	4.19E-04

As shown in table 2, the affinity of the chimeric antibodies ch25D8 and ch25H12 was superior to that of the control antibody ANB020, and the chimeric antibodies had strong binding activity and good drug effect.

Example 2 IL-33/ST2 recombinant protein binding blocking assay

The blocking activity of IL-33 antibodies ch25D8 and ch25H12 was evaluated using recombinant IL-33-his-biotin and ST2-ECD-hFc protein (SEQ ID NO: NP-003847.2 aa1-aa 328) binding assays. Specifically, human ST2 extracellular region recombinant protein (ST 2-hFc) is diluted to 0.5 mu g/mL and 100 mu l/well by PBS, coated with an enzyme link plate and coated overnight at 4 ℃; 5% BSA blocking solution, sealing in a constant temperature incubator at 37 ℃ for 120min, and washing the plate for 3 times by PBST; starting antibodies ch25D8 and ch25H12 to be detected from 30 mug, diluting 8 gradients in a 3-time gradient manner, mixing the diluted 8 gradients with 0.15 mug/ml IL-33-Biotin, adding the mixture into an ELSIA plate, setting multiple holes at each concentration point, uniformly mixing, reacting for 60min at 37 ℃, and washing the plate for 4 times by PBST; adding HRP-anti-streptavidin (Jackson Immuno research) diluted by 1:5000 for reaction for 15min, and washing the plate for 4 times by PBST; and finally adding a TMB substrate for color development, reacting for 15min in a constant-temperature incubator at 37 ℃, stopping the reaction by using 2M HCl, and reading and recording the absorbance of the pore plate with the wavelength of 450 nm. Results as shown in figure 1, ch25D8 was able to dose-dependently inhibit the binding of IL-33 and ST 2.

Example 3 humanization of anti-IL-33 antibodies

The light and heavy chain variable region sequences of the humanized antibody H25D8H1L1 are shown as SEQ ID number 7 and SEQ ID number 8 as follows, and the humanized antibody variable region gene is synthesized, and a humanized antibody recombinant expression vector is constructed according to the method in the example 1, and the antibody is expressed and purified to prepare the humanized antibody. The affinity of the Foretibo assay antibody is shown in table 3.

SEQ ID NO. 7 hzVL1:

DIQLTQSPSSMSASVGDRVTITCRASSSVSYMHWFQQKPGKAPKLWIYRTSNLQSGVPSRFSGSGSGTDYTLTISSMQPEDFATYYCLQRSTYLFMFGQGTKLELK

SEQ ID NO. 8 hzVH1:

QVQLVQSGAEVKKPGASVKLSCKTSGYTFTSYWIQWVRQAPGQGLEWIGEIFPGTGTTYYNQKFQGRATLTTDTSTTTAYMELSSLRSEDTAVYFCARRIRDYYGSNYFDYWGQGTTLTVSS

TABLE 3 humanized antibody affinity assay

Sample	KD (M)	kon(1/Ms)	kdis(1/s)
				ch25D8	6.17E-10	7.66E+05	4.72E-04
h25D8H1L1	6.10E-10	6.00E+05	3.66E-04

Experimental data demonstrate that humanized antibodies retain affinity comparable to that of chimeric antibodies.

Example 4 IL-33 stimulated KU812 cell IL-5 production inhibition assay

IL-33 can stimulate mast cell KU812 (Chinese academy of sciences cell bank, cat. TChu 189) to express IL-5 secretion (Human IL-5 DuoSet ELISA kit (R & D, cat. DY205-05, lot. P151330), the test is used to evaluate the inhibition effect of the anti-IL-33 antibody on IL-33 biological activity. specifically, the final concentration of hIL-33WT is 20ng/ml, the concentrations of a to-be-detected antibody H25D8H1L1 and a control antibody ANB020 are 160 mug/ml, 8 points are diluted by 4 times, 10 middle holes are used, KU812 cells are incubated according to 100000/hole, a 100 mu L system, 24H, one day ahead of time, a capture antibody (240 PBS mu g/ml) is coated, the working solution is diluted to 2 mug/ml, 4 degrees is coated overnight, 50 mu L/hole is sealed by a sealing solution for 1H, three times of washing standard solution 120ng/ml, the 400-fold dilution was diluted to 300pg/ml (1 μ l to 400 μ l), and then diluted 2-fold for 7 points (200 μ l to 200 μ l). And (4) sucking 50 mul of cell culture supernatant and diluted standard solution, adding the supernatant and the diluted standard solution into an ELISA (enzyme-linked immunosorbent assay) pore plate, performing double-hole incubation for 2 hours. Washing for three times, adding the antibody to be detected (7.5 mug/ml stock solution), diluting (1% BSA in PBS) to 125ng/ml working solution, incubating for 2h and washing for three times at 50 mug/hole. And diluting the working solution (1% BSA in PBS) by 40 times to 125ng/ml to obtain SA-HRP (SA-HRP), and incubating for 20-30 min at 50 mu l/hole. And adding 50 mul/hole color development liquid, closing the light for color development for 5-10 min, and adding 100 mul/hole 2M HCl to terminate.

The test results are shown in FIG. 2, and H25D8H1L1 can inhibit the stimulation effect of IL-33 on the expression of IL-5 by KU812 cells in a dose-dependent manner. The IC50 data are shown in table 4, with H25D8H1L1 being significantly better than the control antibody ANB 020.

Table 4. H25D8H1L1 inhibition of IL-33 activity promoting KU812-IL5 production (IC 50: mug/ml)

	h25D8H1L1	ANB020
			IC50（µg/ml）	0.677	5.824

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Sequence listing

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Claims

1. An anti-human IL-33 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein,

the VH comprises the antigenic determinant regions VH CDR1, VH CDR2 and VH CDR3, the VH CDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 14, and the VH CDR2 comprises an amino acid sequence as set forth in EIFPGTGTTYYNX₁KFX₂G, and the VH CDR3 comprises an amino acid sequence shown as SEQ ID NO: 16;

the VL comprises the antigenic determinants VL CDR1, VL CDR2 and VL CDR3, the VL CDR1 comprises as X₃ASSSVSYMH, and the VL CDR2 comprises an amino acid sequence as RTSNLX₄S, and the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13;

wherein the content of the first and second substances,

x1 is E or Q;

x2 is K or Q;

x3 is S or R;

x4 position A or Q.

2. The anti-human IL-33 antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 4, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 3.

3. The anti-human IL-33 antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 8, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 7.

4. The anti-human IL-33 antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

5. The anti-human IL-33 antibody or antigen-binding fragment thereof of claim 4, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody.

6. The anti-human IL-33 antibody or antigen-binding fragment thereof of claim 5, wherein the antibody or antigen-binding fragment thereof is an antibody fragment that specifically binds human IL-33 and is selected from the group consisting of Fv, Fab ', scFv, and F (ab')₂。

7. An isolated polynucleotide encoding an anti-human IL-33 antibody or antigen-binding fragment thereof according to any one of claims 1-6.

8. An isolated polynucleotide encoding the variable region of the light chain or the variable region of the heavy chain, or the light chain or the heavy chain of the anti-human IL-33 antibody or antigen-binding fragment thereof of any one of claims 1-6.

9. A vector comprising one or more polynucleotides of claim 7 or 8.

10. The vector of claim 9, wherein the vector comprises two polynucleotides according to claim 7 or 8, and wherein the vector encodes a VL region and a VH region that together bind IL-33.

11. A vector pair, each vector pair comprising one of the polynucleotides of claim 7 or 8, wherein said vector pairs collectively encode a VL region and a VH region that together bind IL-33.

12. A host cell comprising the polynucleotide of claim 7 or 8 or the vector of claim 9 or 10, or the pair of vectors of claim 11.

13. A composition, comprising:

the anti-human IL-33 antibody or antigen-binding fragment thereof according to any one of claims 1-6, the polynucleotide according to claim 7 or 8, the vector according to claim 9 or 10, the vector pair according to claim 11, or the host cell according to claim 12; and

optionally pharmaceutically acceptable excipients.

14. A method of making an anti-human IL-33 antibody or antigen-binding fragment thereof, comprising:

culturing the host cell of claim 12 under suitable conditions;

separating and recovering the anti-human IL-33 antibody or the antigen-binding fragment thereof.

15. Use of an anti-human IL-33 antibody or antigen-binding fragment thereof according to any one of claims 1-6, the polynucleotide according to claim 7 or 8, the vector according to claim 9 or 10, the vector pair according to claim 11, the host cell according to claim 12, or the composition according to claim 13 for the preparation of a diagnostic agent for inflammatory or cardiovascular diseases.

16. Use of an anti-human IL-33 antibody or antigen-binding fragment thereof according to any one of claims 1-6, the polynucleotide of claim 7 or 8, the vector of claim 9 or 10, the vector pair of claim 11, the host cell of claim 12, or the composition of claim 13 for the manufacture of a medicament for the treatment of inflammation or cardiovascular disease.

17. The use of claim 15 or 16, wherein the inflammation comprises asthma, atopic dermatitis, COPD and chronic obstructive pulmonary disease, chronic sinusitis and rheumatoid arthritis.