CN112480252B - Anti-interleukin-33 antibody and preparation method and application thereof - Google Patents

Abstract

The present invention provides antibodies, antigen-binding fragments thereof, and uses and related methods that bind to IL-33. In particular, the invention provides an antibody and an antigen binding fragment thereof, which specifically bind to human IL-33, nucleic acid molecules encoding the antibody or fragment, vectors and host cells comprising the nucleic acid molecules, methods of making the antibody or fragment, pharmaceutical compositions comprising the antibody or fragment, and methods of treating a condition associated with an allergic disease (including treating atopic dermatitis) or related pharmaceutical uses of the antibody or fragment.

Description

Anti-interleukin-33 antibody, preparation method and application thereof

Technical Field

The present application relates to the field of antibodies, and more particularly, to antibodies against interleukin-33, methods of making, and uses thereof.

Background

Atopic Dermatitis (AD) is a common chronic inflammatory disease characterized by pruritic dermatoses, which is affected in a large proportion (up to 10%) in the adult population of developed countries. There is increasing evidence that it is associated with other allergic diseases such as asthma and food allergies. AD is also part of a process called atopic march, progressing from AD to allergic rhinitis and asthma. Asthma is also the most common respiratory disease, and the body responds to external or internal allergens or non-allergens, and the like, and clinically, the asthma is manifested as repeated paroxysmal chest distress and dyspnea, and severe asthma attacks can even endanger life. Clinical studies have confirmed that Th 2-type cytokines are important mediators in the development of allergic and non-allergic eosinophilic asthma.

Interleukin 33 (IL-33), a cytokine belonging to the IL-1 family that is associated with inflammation. IL-33 induces production of type 2 cytokines, such as IL-4, IL-5 and IL-13, by helper T cells, mast cells, eosinophils and basophils. IL-33 signals by interacting with its receptor ST2 and the IL-1 receptor cofactor (IL 1 RAP) to form a ternary complex. The signaling complex activates intracellular molecules in the NF-. Kappa.beta.and map kinase signaling pathways, thereby mediating their biological effects.

IL-33 is thought to be involved in the development and progression of AD. In inflammatory skin lesions of atopic dermatitis patients, both mRNA and protein of IL-33 were significantly elevated compared to non-inflammatory skin. IL-33 has been shown to be an important molecule for consolidating the function of pathogenic Th2 cells in vivo, and interleukin-33 acts on a series of leukocytes involved in the pathogenesis of atopic diseases. Genetic and functional studies have demonstrated a central role for IL-33 and its receptor ST2 in susceptibility to atopic dermatitis in patients and animal models. IL-33 has been shown in a number of preclinical models to have a critical role when its activity is blocked by pharmacological or genetic means, by modulating a variety of key immune cells in allergic inflammatory diseases, including AD and allergic asthma.

Therefore, the invention aims to develop an antibody which can be combined with IL-33 with high affinity, can effectively neutralize the activity of IL-33 and block IL-33 mediated pathological reaction. Is expected to be used for treating various allergic inflammatory diseases (including AD and asthma).

Disclosure of Invention

The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to human IL-33, nucleic acid molecules encoding the antibodies or fragments, vectors and host cells comprising the nucleic acid molecules, methods of making the antibodies or fragments, pharmaceutical compositions comprising the antibodies or fragments, and methods of treating disorders associated with allergic diseases (including treating atopic dermatitis) or related pharmaceutical uses of the antibodies or fragments.

Antibodies and antigen-binding fragments thereof that bind to human IL-33

In some embodiments, in a first aspect, the present application provides an antibody, or antigen-binding portion thereof, that specifically binds IL-33 comprising a heavy chain variable region comprising an HCDR3 sequence, optionally further comprising an HCDR1 and/or HCDR2 sequence. In some embodiments, the HCDR1 sequence described above comprises a sequence selected from SEQ ID NOs:33 39, 45, 51, 57, 63, 69 and 75. In some embodiments, the HCDR2 sequence comprises a sequence selected from SEQ ID NOs:34 40, 46, 52, 58, 64, 70 and 76. In some embodiments, the HCDR3 sequence comprises an amino acid sequence selected from SEQ ID NOs:35 41, 47, 53, 59, 65, 71 and 77.

In some embodiments, the heavy chain variable region comprises a heavy chain variable region substantially identical to a light chain variable region selected from SEQ ID NOs:2,6, 10, 14, 18, 22, 26 and 30, or the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2,6, 10, 14, 18, 22, 26 and 30.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds IL-33 further comprises a light chain variable region, wherein the light chain variable region comprises an LCDR1, LCDR2, and/or LCDR3 sequence. In certain embodiments, the LCDR1 sequence comprises an amino acid sequence selected from SEQ ID NOs:36 42, 48, 54, 60, 66, 72 and 78. In certain embodiments, the LCDR2 sequence comprises an amino acid sequence selected from SEQ ID NOs:37, 43, 49, 55, 61, 67, 73 and 79. In certain embodiments, the LCDR3 sequence comprises an amino acid sequence selected from SEQ ID NOs:38 44, 50, 56, 62, 68, 74 and 80.

In some embodiments, the light chain variable region comprises a heavy chain variable region substantially identical to a light chain variable region selected from SEQ ID NOs:4,8, 12, 16, 20, 24, 28 and 32, an amino acid sequence having at least 80% homology; or the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs:4,8, 12, 16, 20, 24, 28 and 32.

In some embodiments, the heavy chain of an antibody, or antigen-binding portion thereof, that specifically binds IL-33 comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs:83 89, 95, 101 and 107 or an amino acid sequence having at least 80% homology to the above sequences. Optionally, the light chain of the antibody, or antigen-binding portion thereof, comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:86 92, 98, 104 and 110 or an amino acid sequence having at least 80% homology to the above sequences.

In some embodiments, the antibody that specifically binds IL-33 of the first aspect is a monoclonal antibody.

In some embodiments, the antibody that specifically binds IL-33 of the first aspect is a humanized antibody.

In some embodiments, an IL-33 antibody or antigen-binding portion thereof disclosed herein binds to the same epitope on antibody 20H,127H,177H, 219H,309H or IL-33, or competes with 20H,127H,177H, 219H,309H for binding to IL-33. Wherein the heavy chain sequence of the antibody 20H is as shown in SEQ ID NO: 83. the light chain sequence is shown as SEQ ID NO:86, respectively; and the heavy chain sequence of the antibody 127H is set forth in SEQ ID NO:89 and a light chain sequence is shown as SEQ ID NO:92, respectively; and the heavy chain sequence of the antibody 177H is as set forth in SEQ ID NO:95, and the light chain sequence is shown as SEQ ID NO:98 is shown; and the heavy chain sequence of the antibody 219H is set forth in SEQ ID NO:101, and the light chain sequence is shown as SEQ ID NO:104 is shown; and the heavy chain sequence of the antibody 309H is set forth in SEQ ID NO:107, and the light chain sequence is shown as SEQ ID NO: 110. as shown.

In some embodiments, an antibody or antigen-binding portion thereof disclosed herein is capable of inhibiting IL-5 secretion by KU812 cells.

In a second aspect, the present application provides a nucleotide molecule encoding an antibody or antigen-binding portion thereof that specifically binds IL-33 as described above.

In a third aspect, the present application provides an expression vector comprising a nucleotide molecule as described above.

In some embodiments, the expression vector is pTT5, pUC57, pDR1, pcDNA3.1 (+), pDFFF or pCHO 1.0 or the like.

In a fourth aspect, the present application provides a host cell containing an expression vector as described above. In some embodiments, the host cell is HEK293, COS, CHO, NS0, sf9, sf21, DH5 α, BL21 (DE 3), or TG1, or the like.

In a fifth aspect, the present application provides a method of making an antibody or antigen-binding portion thereof of the first aspect that specifically binds IL-33, the method comprising the steps of:

a) Culturing said host cell of the fourth aspect under expression conditions such that said antibody, or antigen-binding portion thereof, is produced by said host cell, thereby expressing said antibody, or antigen-binding portion thereof; and

b) Isolating and purifying the antibody or antigen-binding portion thereof expressed by a).

In a sixth aspect, the present application provides a pharmaceutical composition comprising the anti-IL-33 antibody, or antigen-binding portion thereof, of the first aspect and a pharmaceutically acceptable carrier.

In some embodiments, the compositions are used to treat IL-33-associated diseases.

In a seventh aspect, the application provides the use of an anti-IL-33 antibody or antigen-binding portion thereof according to the first aspect, or a composition according to the sixth aspect, in the manufacture of a medicament for the prevention or treatment of an IL-33-associated disease, such as an immune-mediated inflammatory response or an inflammatory disease.

The anti-IL-33 antibodies or antigen-binding portions thereof of the present application are capable of specifically binding to IL-33 with one or more of the following effects: blocking the binding of IL-33 to IL-33R; inhibit IL-5 secretion from KU812 cells. The anti-IL-33 antibodies, or antigen-binding portions thereof, of the present application can be used to prevent or treat IL-33-associated diseases, such as immune-mediated inflammatory diseases.

The inventors of the present application have conducted extensive experiments to obtain a group of monoclonal antibodies capable of blocking IL-33 signaling by specifically blocking IL-33 binding to a cell surface IL-33 receptor (IL-33R), which are capable of blocking IL-33 mediated biological activity.

Drawings

FIG. 1 shows the results of measurement of the binding of a mouse-derived anti-human IL-33 monoclonal antibody to human IL-33.

FIG. 2 shows the results of experiments on hIL-33 binding to hIL-33R by the established murine anti-human IL-33 monoclonal antibody.

FIG. 3 shows the results of experiments on the blocking of hIL-33 binding to hIL-33R by a preferred humanized anti-human IL-33 monoclonal antibody.

FIG. 4 shows the results of an experiment in which a preferred humanized anti-human IL-33 monoclonal antibody inhibits IL-5 secretion from KU812 cells.

Detailed Description

The present application provides novel anti-IL-33 antibodies, or antigen-binding portions thereof, that specifically bind to IL-33. In a preferred embodiment, the antibodies of the present application, or antigen binding portions thereof, bind to human IL-33 with high affinity and inhibit the activity of IL-33. Also provided are polynucleotides encoding the antibodies or antigen-binding fragments thereof, vectors comprising the polynucleotides, host cells comprising the polynucleotides or vectors, methods of making and purifying the antibodies, and medical and biological uses of the antibodies or antigen-binding fragments thereof, such as prevention or treatment of IL-33 associated diseases or disorders. Methods of using the antibodies or antigen-binding fragments thereof to detect IL-33 and modulate IL-33 activity are also contemplated.

To facilitate understanding of the present application, certain terms used herein are first defined.

The term "antibody" as used herein refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by a disulfide bond, as well as multimers thereof (e.g., igM). Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH). The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region (abbreviated CL). The light chain constant region comprises one domain (CL 1). The VH and VL regions can be further subdivided into hypervariable regions known as Complementarity Determining Regions (CDRs) into which conserved regions known as Framework Regions (FRs) are interspersed.

As used herein, the term "antigen-binding portion" of an antibody refers to a portion or segment of an intact antibody molecule that is responsible for binding to an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Antigen-binding fragments of antibodies can be prepared from intact antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, among others. Non-limiting examples of antigen-binding moieties include: a Fab fragment; a F (ab') 2 fragment; (ii) a fragment of Fd; (iv) an Fv fragment; single chain Fv (scFv) molecules; a single domain antibody; a dAb fragment and the smallest recognition unit (e.g., an isolated CDR) that consists of amino acid residues that mimic a hypervariable region of an antibody. The term "antigen-binding portion" also includes other engineered molecules such as diabodies, triabodies, tetrabodies, minibodies, and the like.

As used herein, the terms "heavy chain variable region (VH)" and "light chain variable region (VL)" refer to single antibody variable heavy and light chain regions, respectively, that comprise FR1, 2, 3, and 4 and CDR1, 2, and 3.

It is well known to those skilled in the art that the complementarity determining regions (CDRs, usually CDR1, CDR2 and CDR 3) are the regions of the variable region that have the greatest impact on the affinity and specificity of an antibody. There are two common definitions of CDR Sequences for VH or VL, namely the Kabat definition and Chothia definition, see, for example, kabat et al, "Sequences of Proteins of Immunological Interest", national Institutes of Health, bethesda, md. (1991); A1-Lazikani et al, J.mol.biol.273:927-948 (1997); and Martin et al, proc. Natl.acad.sci.usa86:9268-9272 (1989). For a given antibody variable region sequence, can according to Kabat definition or Chothia definition to determine VH and VL sequence in CDR region sequence. In embodiments of the present application, the CDR sequences are defined using Kabat. Herein, CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively; CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.

The sequence of the CDR regions in the variable region sequence can be analyzed in a variety of ways for the variable region sequence of a given antibody, such as can be determined using the online software Abysis (http:// www. Abysis. Org. /).

The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, e.g. binding of an antibody to an epitope of an antigen, e.g. the ability of an antibody to bind to a specific antigen with an affinity at least two times greater than its affinity for a non-specific antigen. It will be appreciated, however, that an antibody is capable of specifically binding to two or more antigens whose sequences are related. For example, an antibody of the invention can specifically bind to IL-33 in humans and non-humans (e.g., non-human primates).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for mutations that may occur naturally in a small number of individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, so long as they exhibit the desired biological activity.

The term "homology", as used herein, is defined as the percentage of residues in an amino acid or nucleotide sequence variant that are identical, if necessary to the maximum percentage, after alignment and the introduction of gaps in the sequence. Methods and computer programs for alignment are well known in the art. "at least 80% homology" as used herein means homology of any value from 80% to 100%, e.g., 85%, 90%, 95%, 99%, etc.

As used herein, the term "IL-33 associated disease" includes diseases and/or symptoms associated with activation of the IL-33 signaling pathway. Exemplary IL-33 related diseases or disorders include immune-mediated inflammatory responses, such as atopic dermatitis, asthma, and the like.

In one aspect, the present application provides an antibody, or antigen-binding portion thereof, that specifically binds IL-33, comprising a heavy chain variable region and/or a light chain variable region. The CDR, VH, VL, heavy and light chain amino acid sequences and corresponding nucleotide sequences suitable for use in the antibodies disclosed herein are exemplified in tables 3-7 below. In certain embodiments, the anti-IL-33 antibody, or antigen-binding portion thereof, comprises an HCDR3, HCDR2, or HCDR1 sequence independently selected from any one of the HCDR3, HCDR2, or HCDR1 sequences shown in table 4. In certain embodiments, an anti-IL-33 antibody of the present application can further comprise a light chain CDR independently selected from any one of the light chain CDR1, CDR2, or CDR3 sequences set forth in table 5. For example, an anti-IL-33 antibody of the present application may comprise any of the heavy chain variable domains shown in tables 3 and 4, optionally paired with any of the light chain variable domains shown in tables 3 and 5.

In some embodiments, the HCDR1 sequence comprises a sequence selected from SEQ ID NOs:33 39, 45, 51, 57, 63, 69 and 75. In some embodiments, the HCDR2 sequence comprises a sequence selected from SEQ ID NOs:34 40, 46, 52, 58, 64, 70 and 76. In some embodiments, the HCDR3 sequence comprises an amino acid sequence selected from SEQ ID NOs:35 41, 47, 53, 59, 65, 71 and 77.

In specific embodiments, the HCDR3 is selected from the group consisting of SEQ ID NOs:35 41, 47, 53, 59, 65, 71 and 77. In a preferred embodiment, the HCDR3 is selected from the group consisting of the amino acid sequences set forth in 47, 71 and 77.

In specific embodiments, the HCDR2 is selected from the group consisting of SEQ ID NOs:34 40, 46, 52, 58, 64, 70 and 76. In a preferred embodiment, the HCDR2 is selected from the group consisting of amino acid sequences as shown at 46, 70 and 76.

In particular embodiments, the HCDR1 is selected from the group consisting of SEQ ID NOs:33 39, 45, 51, 57, 63, 69 and 75. In a preferred embodiment, the HCDR1 is selected from the group consisting of amino acid sequences set forth as 45, 69, and 75.

In some embodiments, an antibody heavy chain variable region disclosed herein comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs:2,6, 10, 14, 18, 22, 26 and 30. In specific embodiments, the heavy chain variable region consists of a sequence selected from SEQ ID NOs:2,6, 10, 14, 18, 22, 26 and 30.

In some embodiments, the amino acid sequence of an antibody heavy chain variable region disclosed herein is identical to SEQ ID NOs:2,6, 10, 14, 18, 22, 26 and 30 have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology. In preferred embodiments, the heavy chain variable region is identical to SEQ ID NOs:88 100 or 106 has a homology of 99% or more.

The antibodies or antigen-binding portions thereof disclosed herein may further comprise a light chain variable region in addition to the heavy chain variable region.

In some embodiments, the LCDR3 of the light chain variable region is selected from SEQ ID NOs:74 and 80, or an amino acid sequence selected from the group consisting of SEQ ID NOs:38 44, 50, 56, 62 and 68. In a preferred embodiment, the LCDR3 is selected from SEQ ID NOs:50 And 74 and 80.

In some embodiments, the LCDR2 is selected from SEQ ID NOs:73 and 79, or an amino acid sequence selected from the group consisting of SEQ ID NOs:37 43, 49, 55, 61 and 67. In a preferred embodiment, the LCDR2 is selected from SEQ ID NOs:49 73 and 79.

In some embodiments, LCDR1 is selected from SEQ ID NOs:72 and 78, or an amino acid sequence selected from SEQ ID NOs:36 42, 48, 54, 60 and 66. In a preferred embodiment, LCDR1 is selected from SEQ ID NOs:48 72 and 78, respectively.

In some embodiments, the antibody light chain variable region disclosed herein comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs:4,8, 12, 16, 20, 24, 28 and 32. In specific embodiments, the light chain variable region is encoded by a sequence selected from the group consisting of SEQ ID NOs:4,8, 12, 16, 20, 24, 28 and 32.

In some embodiments, the amino acid sequence of the antibody light chain variable region disclosed herein is identical to SEQ ID NOs:4,8, 12, 16, 20, 24, 28 or 32 has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology. In a preferred embodiment, the light chain variable region is substantially identical to SEQ ID NOs:91 103 or 109 has over 99% homology.

In some embodiments, the heavy or heavy chain variable region, the light chain or the light chain variable region of the antibodies disclosed herein can be substituted, deleted or added with at least one amino acid based on the respective corresponding specific amino acid sequences listed above, and the resulting mutants still retain the activity of binding IL-33.

In certain embodiments, the number of amino acid substitutions, deletions or additions is 1 to 30, preferably 1 to 20, more preferably 1 to 10. In preferred embodiments, the sequence variant differs from the original amino acid sequence by substitutions, deletions and/or additions of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In more preferred embodiments, the sequence variant differs from the original amino acid sequence by a substitution, deletion, or addition of about 1, 2, 3, 4, or 5 amino acids. In particular embodiments, the amino acid substitution is a conservative substitution.

In a preferred embodiment, the antibody disclosed herein is antibody 127h,219h or 309H. Wherein the heavy chain sequence of the antibody 127H is shown as SEQ ID NO:89 and the light chain sequence is shown as SEQ ID NO:92, respectively; and the heavy chain sequence of the antibody 219H is set forth in SEQ ID NO:101, and the light chain sequence is shown as SEQ ID NO:104 is shown; and the heavy chain sequence of the antibody 309H is set forth in SEQ ID NO:107, and the light chain sequence is shown as SEQ ID NO: 110.

In some embodiments, the antibodies disclosed herein, or antigen binding portions thereof, bind to the same epitope on interleukin-33 as antibodies 20H,127H,177H, 219H,309H, or compete with antibodies 20H,127H,177H, 219H,309H for binding to interleukin-33.

In some embodiments, the antibodies disclosed herein are monoclonal antibodies. In particular embodiments, the antibodies disclosed herein are humanized antibodies.

The antibodies, or antigen-binding portions thereof, disclosed herein are capable of specifically binding IL-33. In specific embodiments, the antibody or antigen binding portion thereof specifically binds to human IL-33 or monkey IL-33. In a preferred embodiment, the antibody, or antigen binding portion thereof, specifically binds to human IL-33.

In some embodiments, an antibody or antigen-binding portion thereof disclosed herein is capable of inhibiting IL-5 secretion by KU 812.

For example, the inventors of the present application performed in vitro and in vivo biological experiments on the anti-human IL-33 monoclonal antibody disclosed herein, and the results showed that the antibody binds well to IL-33.

Specifically, the inventors of the present application carried out experiments such as binding detection, experimental analysis for blocking the binding of IL-33 to IL-33R, and in vitro cell function detection of an anti-human IL-33 monoclonal antibody. The experimental results show that the anti-human IL-33 monoclonal antibody disclosed by the invention can be combined with IL-33, block the signal conduction between IL-33 and IL-33R and inhibit the generation of inflammatory reaction.

The present application also provides nucleotide molecules encoding the antibodies disclosed herein, or antigen-binding portions thereof, vectors comprising the polynucleotides, host cells comprising the polynucleotides or vectors, and methods of making and purifying the antibodies.

In some embodiments, the nucleotide molecule encoding the antibody, or antigen-binding portion thereof, is operably linked to a control sequence that is recognized by a host cell transformed with the vector.

In some embodiments, any suitable expression vector may be used in the present application. For example, the expression vector may be one of pTT5, pUC57, pDR1, pcDNA3.1 (+), pDFFF and pCHO 1.0. Expression vectors may include fusion DNA sequences with appropriate transcriptional and translational regulatory sequences attached.

In some embodiments, useful host cells are cells containing the above-described expression vectors, which may be eukaryotic cells, such as mammalian or insect host cell culture systems, can be used for expression of the antibodies or antigen-binding portions thereof of the present application. For example, HEK293 cells, COS, CHO, NS0, sf9, sf21, etc. can be used in the present invention. The host cell may be a prokaryotic cell containing the above expression vector, and may be, for example, DH 5. Alpha., BL21 (DE 3), TG1 or the like.

In some embodiments, the methods of preparing an anti-human IL-33 monoclonal antibody disclosed herein comprise: culturing the host cell under expression conditions, thereby expressing the anti-human IL-33 monoclonal antibody; isolating and purifying the expressed anti-human IL-33 monoclonal antibody. Using the above method, the recombinant protein can be purified as a substantially homogeneous substance, for example, as a single band on SDS-PAGE electrophoresis.

In some embodiments, the anti-IL-33 antibodies disclosed herein can be isolated and purified using affinity chromatography, and the anti-IL-33 antibodies bound to the affinity column can be eluted using conventional methods, such as high salt buffers, pH changes, and the like, depending on the nature of the affinity column being used.

In some embodiments, the humanized anti-human IL-33 monoclonal antibodies disclosed herein are obtained by the following method: IL-33 antigen prepared in a laboratory is used for immunizing a Balb/c mouse, and spleen cells of the mouse are taken to be fused with hybridoma cells after the titer of the immunized mouse is higher for multiple times, and hybridoma cell strains with the IL-33 inhibition functional activity are screened out. More specifically, the inventors of the present invention have made extensive experiments to express IL-33 antigen and IL-33R separately, and then have immunized mice with IL-33 antigen mixed with different adjuvants, and then further fused splenocytes of the mice with hybridoma cell line sp2/0, and then screened positive cell lines using IL-33 antigen from the fused hybridomas, and have verified that the binding of IL-33 to IL-33R is blocked and the function of IL-33 is surely inhibited, to obtain target cell lines. After the target molecule is subjected to humanization transformation, the light chain and heavy chain genes are simultaneously cloned into a eukaryotic expression vector pTT5. The expression vector is used for producing the antibody by transient transfection of HEK293 cells and serum-free medium culture, and humanized anti-human IL-33 monoclonal antibody is separated or purified by a Protein A affinity column.

In other embodiments, the parent antibody of murine origin may be further altered using techniques conventional in the art, such as PCR mutagenesis, to produce chimeric or humanized or other variant forms of the antibody. Parent antibodies of the present application can be mutagenized, for example, within the Complementarity Determining Regions (CDR) domains of an antigen to produce variant antibodies, which can be screened for the presence of properties of interest, such as binding affinity (lower KD), IC50, specificity, preferential binding, and the like. Preferably, the property of interest in the variant antibody is an improvement over the property in the parent antibody. Amino acid substitution variant antibodies are preferred, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues of the parent antibody molecule are removed and a different residue is inserted in its position. The most interesting site for substitution mutagenesis is one or more CDR regions, but Framework Region (FR) alterations are also contemplated. Conservative amino acid substitutions are preferred, and non-conservative amino acid changes can also be introduced and the resulting variant antibodies screened for properties of interest.

The application also provides the use of an anti-IL-33 antibody, or a composition comprising an anti-IL-33 antibody, in the manufacture of a medicament for the prophylaxis or treatment of an IL-33 associated disease or condition. In some embodiments, the IL-33-associated disease or condition is an immune-mediated inflammatory response or an immune-mediated inflammatory disease.

In some embodiments, the anti-IL-33 antibodies disclosed herein may be used as anti-immune mediated inflammatory response agents. An anti-immune-mediated inflammatory response agent, as referred to herein, refers to an agent that inhibits and/or treats immune-mediated inflammatory responses, e.g., it may delay the development of and/or reduce the severity of symptoms associated with immune-mediated inflammatory responses. In some embodiments, the medicament may reduce an existing inflammatory response with symptoms and prevent the appearance of other symptoms. In some embodiments, the medicament may also reduce or prevent the transfer of an inflammatory response.

In this specification and claims, the words "comprise", "comprises", and "comprising" mean "including but not limited to", and are not intended to exclude other moieties, additives, components, or steps.

It should be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example of the present application may be applied to any other aspect, embodiment or example described herein unless incompatible therewith.

The foregoing disclosure generally describes the present application and the following examples are presented to further illustrate the present application and are not to be construed as limiting the present application. The examples do not include detailed descriptions of conventional methods such as those for constructing vectors and plasmids, methods for inserting genes encoding proteins into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in numerous publications.

Examples

The present disclosure is further described below in conjunction with the following examples, which are not intended to limit the scope of the present disclosure. Experimental procedures for conditions not specified in the examples are generally carried out according to conventional conditions, or according to conditions recommended by manufacturers of raw materials or commercial products (see Sambrook et al, molecular cloning, A laboratory Manual, cold spring harbor laboratory; ausubel et al, contemporary methods of molecular biology, greene Press Association, wiley Interscience, N.Y.). Reagents of specific sources are not indicated, and are conventional reagents purchased in the market.

Example 1Preparation of human IL-33 antigen, human IL-33R extracellular protein and reference antibody pf158

The human IL-33 antigen sequence was from UniProt (UniProtKB-O95760), codon-optimized by Probiotics according to the codon usage bias of Cricetulus griseus, and amino acid fragments 112-270 were synthesized and cloned into pET-28a (+) vector (from Probiotics) to obtain pET-hIL-33-His. Using pET-hIL-33-His as a template, respectively inserting a His tag (HHHHHHHHHH) and a Flag tag (DYKDDDDK) into the N end and the C end of the hIL-33 fragment by a PCR method to respectively obtain N-His-hIL-33 and hIL-33-Flag fragments, constructing the fragments on a pET-28a (+) expression vector, verifying sequencing, and selecting a clone with a completely correct sequence for prokaryotic expression production. Proteins were purified from E.coli supernatants using nickel columns (from GE) and Flag affinity chromatography (from Sigma) for immunization and further analysis and study of mice as follows.

The human IL-33R sequence was from UniProt (UniProtKB-Q01638), and the cloning vector for this gene was purchased from Chinesia. The vector is used as a template, and the hFc fragment and Flag tag (DYKDDDDK) are respectively inserted into the C end of the extracellular segment (amino acids 19-328) of hIL-33R by a PCR method to obtain the hIL-33R-ECD-hfc and the hIL-33R-ECD-Flag fragments. And recombining the hIL-33R-ECD-hfc and hIL-33R-ECD-flag fragments to connect the hIL-33R-ECD-flag fragments into the pTT5 vector, verifying sequencing, and selecting a clone with a completely correct sequence for eukaryotic expression production. hIL-33R-ECD-hfc-pTT5 and hIL-33R-ECD-flag-pTT5 vectors were transfected into HEK293E cell lines (stored in the laboratory) by the PEI method, respectively. After 5 days of culture in Freestyle293 medium (from Gibco) containing 3mM valproic acid, the protein was purified from the cell culture supernatant by protein a (from GE) and Flag affinity chromatography (from Sigma) for further analysis and study as follows.

The amino acid sequence of the reference antibody pf158 is from patent WO2017187307A1. IgG1 was used as the constant region of the antibody. After codon optimization, nucleotide sequences were synthesized from the whole gene, and the sequences were subcloned into pUC57 vector (from Biotech), to obtain pUC57-pf158-VH, pUC57-pf158-VL, pUC57-IgG1-CH, and pUC57-IgG1-CL. The variable regions VH and VL of pf158 are spliced with IgG1-CH and IgG1-CL by a PCR method to obtain pf158-HC and pf158-LC fragments which are cloned to a pTT5 expression vector, and the correct cloning vectors marked as pf158-HC-pTT5 and pf158-LC-pTT5 are obtained by sequencing verification. Both vectors were transiently transfected into a HEK293E cell line, and after 5 days of culture in Freestyle293 medium containing 3mM valproic acid, the pf158 antibody Protein was purified from the cell culture supernatant using a Protein a affinity column (purchased from GE).

Example 2Human IL-33 antigen immunized mice

The hIL-33-his antigen (at a dose of 100. Mu.g/mouse) was diluted to 75. Mu.l with physiological saline, mixed with Freund's complete adjuvant of equal volume, and subjected to ultrasonic emulsification to complete the mixture, followed by subcutaneous multi-injection into 4-5-week-old Balb/c mice (purchased from Shanghai Ling-Biotech Co., ltd., animal production license number: SCXK 2013-0018). Three weeks later, the same antigen (at a dose of 50. Mu.g/mouse) was diluted to 75. Mu.l with the same physiological saline and mixed with an equal volume of Freund's incomplete adjuvant, and after completion of the ultrasonication, the mice were subjected to subcutaneous multiple immunizations, and these immunizations were repeated two weeks later. All mice were tail trimmed one week after the third immunization, bled and sera were isolated and tested for serum titer using ELISA coated with hIL-33-his antigen. For mice with serum antibody titers > 10000, shock immunization was performed one week after blood draw: tail vein injection of 10 u g antigen protein/100 u l normal saline/mouse.

The detection of the titres was carried out by ELISA: ELISA plates were coated with hIL-33-his antigen at a concentration of 1. Mu.g/ml, 100. Mu.l per well, overnight at 4 ℃. PBST (PBS containing 0.5% Tween-20) washed the plates 2 times and patted dry. Each well was blocked with 200. Mu.l of a coating solution containing 1% BSA at room temperature for 4 hours, then patted dry, and stored in a freezer at-20 ℃ until use. In the detection process, 100 mu l of mouse serum with different concentrations is added into each hole of an ELISA plate, 2 multiple holes are arranged, and the mixture is incubated for 1.5 hours at room temperature. PBST was washed 3 times and then patted dry. Mu.l of HRP-labeled rabbit anti-mouse Ig antibody (purchased from Sigma) diluted 1: 10000 times in PBST was added and incubated at room temperature for 1 hour. PBST was washed 3 times and then patted dry. Adding 100 μ l of developing solution into each well (mixing ELISA developing solution A and developing solution B at a volume ratio of 1: 1 before use) for developing, and adding 100 μ l of 2M H into each well ₂ SO ₄ The reaction was terminated by the stop solution. Immediately, the OD value of each well was measured with a microplate reader (Molecular Device) at a wavelength of 450 nm.

Example 3Hybridoma cell fusion and screening

Splenocytes were taken three days after the mice were shock immunized for fusion.

Taking well-grown hybridoma sp2/0 cells (from cell bank of the culture Collection of type culture Collection of Chinese academy of sciences, accession number TCM-18) at 37 deg.C and 5% CO ₂ Culturing in incubator, and changing liquid one day before fusion. The fusion and screening process was as follows: spleen of the mice is taken, ground, washed and counted. Spleen cells and sp2/0 cells were mixed at a ratio of 10: 1 and centrifuged at 1500rpm for 7 minutes. The supernatant was washed off. 1ml of PEG (1450) was added within 1 minute, the reaction mixture was gently shaken for 90 seconds, 5ml of serum-free DMEM (purchased from Gibco) medium was added within 2.5 minutes, and 5ml of serum-free DMEM medium was added once again to terminate the reaction, and the reaction mixture was allowed to stand for 5 minutes and centrifuged at 1280rpm for 8 minutes. Cells were seeded evenly into 96-well plates at 200. Mu.l per well, in the number of two million sp2/0 cells per 96-well plate. The culture medium is firstly screened by HAT culture medium containing hypoxanthine (H), methotrexate (A) and thymidine (T), the liquid is changed by half every 3-4 days, and the culture medium is changed by HT on the 10 th day. After 10 days, when the hybridoma cells are more than 10% of the bottom of the 96-well plate, taking the supernatant and carrying out ELISA detection by using an ELISA plate coated by hIL-33-his antigen. The ELISA detection method was the same as described in example 2. Meanwhile, the antibody is subjected to secondary screening by using the hIL-33-flag by using the same ELISA detection method so as to remove the anti-His positive antibody. The hu-IL-33 positive hybridoma clones were selected for expansion in 24-well plates and subcloned by limiting dilution. Obtaining the hybridoma strain which stably expresses the target antibody, and then preserving and building a library.

Example 4Mouse anti-human IL-33 monoclonal antibody blocks the combination of human IL-33 and IL-33R extracellular region protein

The blocking of IL-33 binding to hIL-33R-ECD-hfc by murine anti-human hIL-33 monoclonal antibodies was investigated by ELISA. The hIL-33-his antigen is coated on the enzyme label plate, after being closed, hIL-33R-ECD-hfc and 300 mul of rat-derived anti-human hIL-33 monoclonal antibody hybridoma cell culture supernatant in subclone are added simultaneously, and finally HRP antibody is added for color development detection. The cell line capable of blocking hIL-33 and hIL-33R-ECD-hfc binding was retained for the next round of subcloning.

A preferred murine anti-human IL-33 monoclonal antibody was affinity purified by Protein G affinity chromatography and then quantified by BCA method (reagents from Thermo). The blocking of IL-33 binding to hIL-33R-ECD-hfc by murine anti-human hIL-33 monoclonal antibodies was investigated by ELISA. 307 monoclonal antibodies were analyzedFig. 1 shows representative experimental results. Table 1 lists the IC's of partially preferred antibodies ₅₀ Data, IC ₅₀ All are below 63 ng/ml.

Table 1: exemplary murine anti-human IL-33 monoclonal antibodies block hIL-33 binding to hIL-33R-ECD-hfc

Antibody numbering	IC 50 (ng/ml)
		5	61.53
20	41.15
		76	43.79
96	39.08
		97	39.98
113	39.04
		119	39.12
127	37.74
		130	33.17
158	45.99
		174	49.64
177	62.66
		205	50.21
209	50.2
		219	61.33
223	52.18
		258	49.12
260	51.92
		309	30.23

Example 5Affinity detection of murine anti-human IL-33 monoclonal antibody binding to human IL-33

A preferred murine anti-human IL-33 monoclonal antibody was affinity purified by Protein G affinity chromatography and then quantified by BCA method. Conjugation of anti-human IL-33 monoclonal antibody to hIL-33-hisSynthetic EC ₅₀ Detection was performed by ELISA. The detection method was as in example 3.1 u g/ml hIL-33-his antigen coated ELISA plate, adding different concentrations of murine anti-human IL-33 monoclonal antibody for detection.

307 monoclonal antibodies were analyzed, and FIG. 2 shows representative experimental results. Table 2 lists the EC for some preferred antibodies ₅₀ Data showing that these antibodies have a higher affinity for human IL-33, EC ₅₀ All are below 66 ng/ml.

Table 2: exemplary murine anti-human hIL-33 monoclonal antibodies have affinity for human IL-33-his

Antibody numbering	EC 50 (ng/ml)
		5	36.58
20	41.08
		76	51.98
96	57.38
		97	65.11
113	55.90
		119	56.51
127	43.92
		130	45.57
158	49.51
		174	50.81
177	44.37
		205	40.42
209	33.25
		219	35.69
223	45.66
		258	41.54
260	39.88
		309	52.31

Example 6Determination of murine anti-human IL-33 monoclonal antibody sequence

Total RNA of each hybridoma cell line was extracted using Trizol (purchased from Shanghai Bionical Co.), mRNA was reverse-transcribed into cDNA using a reverse transcription kit (purchased from Thermo Co.), the light chain variable region and heavy chain variable region genes of Mouse-derived anti-human hIL-33 monoclonal antibody were amplified by PCR using Mouse Ig-Primer Set (purchased from Novagen Co.) as a Primer, and then the PCR products were cloned into pMD18-T vector, sequenced and analyzed for variable region gene sequences. According to the results of various functional experiments and early druggability analysis, 8 antibodies shown in the table 3 are finally selected as lead antibodies, and the nucleotide sequences of the light chain variable regions of the antibodies are obtained by sequencing. The amino acid sequences obtained by transformation were aligned in GenBank and all sequences were consistent with the characteristics of the mouse IgG variable region genes. The amino acid sequences of the light chain variable region and the heavy chain variable region of the murine anti-human IL-33 monoclonal antibody were analyzed according to the Kabat's rule and 3 CDRs were determined, as detailed in tables 4 and 5.

Table 3: nucleotide sequence and amino acid sequence of mouse IL-33 antibody heavy chain variable region and light chain variable region

Table 4: heavy chain CDR amino acid sequence of murine IL-33 antibody

Table 5: light chain CDR amino acid sequence of murine IL-33 antibody

Example 7Humanization of murine anti-human IL-33 monoclonal antibodies

Based on the results of sequence analysis, antibodies 20, 127, 177, 219 and 309 were picked and used for the construction of chimeric antibodies and humanized antibodies, and the amino acid sequences of the humanized antibodies are shown in tables 6 and 7. The chimeric antibody is constructed by intercepting the heavy chain variable region and the light chain variable region of a murine antibody and connecting the heavy chain variable region and the light chain variable region with the light chain constant region of human IgG1 respectively by overlapping PCR.

The amino acid sequences of the light chain variable region and the heavy chain variable region of an anti-human IL-33 monoclonal antibody of murine origin were analyzed according to the Kabat's rule and 3 CDRs and 4 FRs were determined. In the case of the antibody No. 309, heavy chain CDR-grafted antibodies were constructed by performing homology comparison between NCBI IgBlast and human IgG Germline sequences (Germinne), selecting IGHV 1-46X 01 as a heavy chain CDR-grafted template, and grafting the heavy chain CDR region of the murine anti-human IL-33 monoclonal antibody 309 into the IGHV 1-46X 01 framework region. Similarly, by comparing the sequence homology with human IgG germ line, IGKV1-39 x 01 was selected as a light chain CDR-grafted template, and the light chain CDR region of a murine anti-human IL-33 monoclonal antibody 309 was grafted into the framework region of IGKV1-39 x 01 to construct a CDR-grafted antibody of the light chain, which was defined as 309-Gr (309-Grafting). Meanwhile, on the basis, the amino acid sites of some framework regions are subjected to back mutation. In the case of back-mutation, the amino acid sequence is Kabat-encoded and the position of the site is indicated by the Kabat code. Preferably, for the heavy chain variable region, M at position 48 is reverted to I, M at position 70 is reverted to L, R at position 72 is reverted to V, T at position 74 is reverted to K, S at position 77 is reverted to N, and V at position 79 is reverted to a; for the light chain variable region sequences, L at position 46 was returned to V and Y at position 49 was returned to S (both above heavy and light chain encodings are based on Kabat). The above-mentioned variable region gene sequence was codon-optimized and synthesized by the organism according to the codon usage preference of Cricetulus griseus. The synthetic humanized variable region sequences were ligated to human IgG1 constant regions, which was defined as a humanized antibody of antibody No. 309 (309-Humanization, 309H).

The remaining 5 antibodies were also humanized using the same principles described above. Transient expression vectors of humanized heavy chains and light chains are respectively constructed by utilizing pTT5 vectors, and the light chains and the heavy chains are combined to carry out transient transfection and express antibodies by utilizing a HEK293 system. HEK293 cells in Free Style293 Expression Medium (purchased from Gibco company) Medium culture, using PEI transfection method to plasmid transfer into cells for 5 days after cell supernatant, using Protein A purification to obtain antibody.

Table 6: nucleotide sequence and amino acid sequence of heavy chain variable region and light chain variable region of humanized anti-IL-33 antibody

Table 7: amino acid sequences of heavy and light chains of exemplary humanized anti-IL-33 antibodies

Example 8Humanized anti-human IL-33 monoclonal antibody blocks the combination of human IL-33 and extracellular region protein

The humanized anti-human IL-33 monoclonal antibody was affinity-purified by Protein A affinity column, and then quantified by BCA method. Blocking of IL-33 binding to hIL-33R-ECD-hfc by humanized anti-human hIL-33 monoclonal antibodies was studied by ELISA. The detection method was as in example 4. FIG. 3 shows representative experimental results, and Table 8 lists the IC of partial antibodies ₅₀ And (6) data. The experimental result shows that the blocking capability of the humanized anti-human IL-33 monoclonal antibody on hIL-33 binding to hIL-33R-ECD-hfc is obviously higher than that of the reference antibody pf158.

Table 8: humanized antibody pair hIL-33 binding hIL-33R-ECD-hfc positive cleavage

Antibody numbering	IC 50 (ng/ml)
		pf158	139.60
127H	49.95
		219H	93.53
309H	65.93

Example 9Humanized anti-human IL-33 monoclonal antibody inhibits the secretion of IL-5 by KU812 cells

Taking KU812 cells (ATCC, CRL-2099) with good growth state, counting, and re-suspending with recombinant hIL-33 at a final concentration of 50ng/ml to 5x10 ⁵ Cell suspension/ml. The medium was RPMI1640 medium (purchased from Gibco), which contains 10% fetal bovine serum (purchased from Sigma), 100U/ml penicillin (purchased from Gibco) and 100mg/ml streptomycin (purchased from Gibco), and was called RPMI-1640 complete medium. 37 ℃ and 5% CO ₂ After 0.5 hour incubation, different concentrations of antibody were added. Different concentrations of humanized anti-human IL-33 monoclonal antibody (250. Mu.g/ml, 3-fold dilution, 9 different concentrations) were diluted with medium solution, 100. Mu.l per well, added to a 96-well flat-bottom cell culture plate (purchased from Corning), and then 100. Mu.l of cell suspension was added per well. Each group was set up 2 replicate wells, 37 ℃,5% CO ₂ And (5) performing medium incubation for 24 hours. Taking supernatant and pressing IL-5 detection kit (R)&D, cat: S5000B) instructions for the operation of detecting IL-5 secretion, using a microplate reader (Molecular Device) to measure the OD value of each well at a wavelength of 450 nm.

The humanized anti-human IL-33 monoclonal antibody was analyzed for the inhibitory activity of KU812 to secrete IL-5 (FIG. 4), and the data of 3 antibodies 127H,219H, 309H are shown in Table 9. The result shows that 127H and 309H have stronger inhibition effect on the IL-5 secretion of KU812 cells, and the functional activity of the antibody is obviously superior to that of a reference antibody pf158.

Table 9: inhibition of IL-5 secretion by KU812 by different humanized antibodies

Antibody numbering	IC 50 (ng/ml)
		pf158	658.00
127H	108.00
		219H	2261.00
309H	16.45

It is to be understood that the present disclosure, although illustrated in some form, is not limited to what is shown and described herein. It will be apparent to those skilled in the art that various changes can be made in the embodiments and/or in a feature or parameter without departing from the scope of the application. Such variations are intended to be within the scope of the present disclosure.

Claims (12)

1. An antibody or antigen-binding portion thereof that specifically binds interleukin-33, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region,

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 10; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 12;

or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 88; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 91.

2. The antibody or antigen-binding portion thereof of claim 1, wherein the antibody has a heavy chain sequence as set forth in SEQ ID NO. 89 and a light chain sequence as set forth in SEQ ID NO. 92.

3. The antibody or antigen-binding portion thereof of claim 1 or 2, wherein the antibody or antigen-binding portion thereof specifically binds to human interleukin-33 or monkey interleukin-33.

4. The antibody or antigen-binding portion thereof of claim 1 or 2, wherein the antibody or antigen-binding portion thereof is capable of inhibiting IL-5 secretion by KU812 cells.

5. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of any one of claims 1-4 and a pharmaceutically acceptable carrier.

6. A nucleotide molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1-4.

7. An expression vector comprising the nucleotide molecule of claim 6.

8. Host cell, characterized in that it comprises a nucleotide molecule according to claim 6 or an expression vector according to claim 7.

9. A method of producing the antibody, or antigen-binding portion thereof, of any one of claims 1-4, comprising:

a) Culturing the host cell of claim 8 under expression conditions such that the host cell is capable of producing the antibody, or antigen-binding portion thereof, thereby expressing the antibody, or antigen-binding portion thereof; and

b) Isolating and purifying the antibody or antigen-binding portion thereof expressed in step a).

10. Use of the antibody or antigen-binding portion thereof of any one of claims 1-4, or the pharmaceutical composition of claim 5, in the manufacture of a medicament for preventing or treating an IL-33-associated disease.

11. The use according to claim 10, wherein the IL-33 related disease is an immune-mediated inflammatory disease.

12. The use according to claim 11, wherein the immune-mediated inflammatory disease is atopic dermatitis or asthma.

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