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

Abstract

The present invention provides antibodies that bind IL-33, antigen-binding fragments thereof, uses and related methods. Specifically, the present invention provides a specific binding human IL-33 antibody and its antigen-binding fragment, a nucleic acid molecule encoding the antibody or fragment, a vector comprising the nucleic acid molecule and a host cell, and a method for preparing the antibody or fragment , a pharmaceutical composition comprising the antibody or fragment, and a method for treating a disease related to an allergic disease (including treating atopic dermatitis) or a related pharmaceutical use of the antibody or fragment.

Description

Anti-interleukin-33 antibody and its preparation method and application

technical field

The present application relates to the field of antibodies, more specifically, the present application relates to anti-interleukin-33 antibodies and their preparation methods and applications.

Background of the invention

Atopic dermatitis (AD) is a common chronic inflammatory disease characterized by pruritic skin lesions that affects a large proportion (up to 10%) of the adult population in developed countries. Mounting evidence links it to other allergic conditions such as asthma and food allergies. AD is also part of a process called atopic march, from which AD progresses to allergic rhinitis and asthma. Asthma is also one of the most common respiratory diseases. The body responds to external or internal allergens or non-allergens. It is clinically manifested as repeated paroxysmal chest tightness and dyspnea, and severe asthma attacks can even be life-threatening. Clinical studies have confirmed that Th2 cytokines are important mediators for the occurrence and development of allergic and non-allergic eosinophilic asthma.

Interleukin 33 (IL-33) is an inflammation-related cytokine belonging to the IL-1 family. IL-33 induces the 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 interacts with its receptor ST2 and IL-1 receptor accessory protein (IL1RAP) to form a ternary complex for signal transduction. This signaling complex mediates biological effects by activating intracellular molecules in the NF-κβ and map kinase signaling pathways.

IL-33 is considered to be related to the occurrence and development of AD. Both mRNA and protein of IL-33 were significantly elevated in inflamed skin lesions of patients with atopic dermatitis compared with non-inflamed skin. IL-33 has been proved to be an important molecule to consolidate the function of pathogenic Th2 cells in vivo, and interleukin-33 can act on a series of leukocytes related to 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 to have a critical role in many preclinical models when its activity is blocked by pharmacological or genetic approaches by modulating various key immune cells in allergic inflammatory diseases, including AD and allergic asthma .

Therefore, the present invention aims to develop antibodies binding to IL-33 with high affinity, which can effectively neutralize the activity of IL-33 and block the pathological response mediated by IL-33. It is expected to be used in the treatment of various allergic inflammatory diseases (including AD and asthma).

Contents of the invention

The present disclosure provides an antibody specifically binding to human IL-33 and its antigen-binding fragment, a nucleic acid molecule encoding the antibody or fragment, a vector and a host cell comprising the nucleic acid molecule, and a method for preparing the antibody or fragment, comprising the Pharmaceutical compositions of antibodies or fragments, and methods or related pharmaceutical uses of antibodies or fragments for treating conditions associated with allergic diseases, including treating atopic dermatitis.

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

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

In some embodiments, the heavy chain variable region described above comprises an amino acid sequence having at least 80% homology to an amino acid sequence selected from the group consisting of 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 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 LCDR1, LCDR2 and/or LCDR3 sequences. In certain embodiments, the LCDR1 sequence comprises an amino acid sequence selected from the group consisting of 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 the group consisting of 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 the group consisting of SEQ ID NOs: 38, 44, 50, 56, 62, 68, 74 and 80.

In some embodiments, the light chain variable region described above comprises an amino acid sequence having at least 80% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28 and 32; or The light chain variable region comprises an amino acid sequence selected from 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 an amino acid sequence selected from SEQ ID NOs: 83, 89, 95, 101, and 107 or has at least 80% identity to the aforementioned sequences. source amino acid sequence. Optionally, the light chain of the antibody or antigen-binding portion thereof comprises an amino acid sequence selected from SEQ ID NOs: 86, 92, 98, 104 and 110 or an amino acid sequence having at least 80% homology to the aforementioned sequences.

In some embodiments, the antibody specifically binding to IL-33 described in the first aspect is a monoclonal antibody.

In some embodiments, the antibody specifically binding to IL-33 described in 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 IL-33 as antibody 20H, 127H, 177H, 219H, 309H, or binds to 20H, 127H, 177H, 219H, 309H competes for binding to IL-33. Wherein the heavy chain sequence of the antibody 20H is shown in SEQ ID NO: 83, the light chain sequence is shown in SEQ ID NO: 86; and the heavy chain sequence of the antibody 127H is shown in SEQ ID NO: 89, the light chain sequence The sequence is shown in SEQ ID NO: 92; and the heavy chain sequence of the antibody 177H is shown in SEQ ID NO: 95, and the light chain sequence is shown in SEQ ID NO: 98; and the heavy chain sequence of the antibody 219H is shown in SEQ ID NO: 101, the light chain sequence is shown in SEQ ID NO: 104; and the heavy chain sequence of the antibody 309H is shown in SEQ ID NO: 107, the light chain sequence is shown in SEQ ID NO: 110.

In some embodiments, an antibody disclosed herein, or an antigen-binding portion thereof, 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 an antigen-binding portion thereof that specifically binds IL-33 as described above.

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

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

In a fourth aspect, the present application provides a host cell containing the expression vector as described above. In some embodiments, the host cell is HEK293, COS, CHO, NSO, sf9, sf21, DH5α, BL21(DE3) or TG1, etc.

In a fifth aspect, the present application provides a method for preparing the antibody or antigen-binding portion thereof that specifically binds IL-33 described in the first aspect, the method comprising the following steps:

a) culturing the host cell of the fourth aspect under expression conditions that enable the host cell to produce the antibody or antigen-binding portion thereof, thereby expressing the antibody or antigen-binding portion thereof; and

b) isolating and purifying said antibody or antigen-binding portion thereof expressed in a).

In a sixth aspect, the present application provides a pharmaceutical composition, which comprises the anti-IL-33 antibody or its antigen-binding portion described in the first aspect and a pharmaceutically acceptable carrier.

In some embodiments, the composition is used to treat IL-33-related diseases.

In the seventh aspect, the present application provides the anti-IL-33 antibody or its antigen-binding portion described in the first aspect, or the composition described in the sixth aspect for preventing or treating IL-33-related diseases, such as immune-mediated Inflammation-induced inflammatory response or drug application in inflammatory diseases.

The anti-IL-33 antibody or antigen-binding portion thereof of the present application can specifically bind to IL-33, and has one or more of the following effects: blocking the binding of IL-33 to IL-33R; inhibiting the IL-33 expression of KU812 cells; 5 secretion. The anti-IL-33 antibody or antigen-binding portion thereof of the present application can be used to prevent or treat IL-33-related diseases, such as immune-mediated inflammatory diseases.

The inventors of the present application have conducted a large number of experiments and obtained a group of monoclonal antibodies that can block IL33 signal transduction by specifically blocking the binding of IL-33 to the cell surface IL-33 receptor (IL-33R), which can Block IL-33-mediated biological activity.

Description of drawings

Fig. 1 is the measurement result of the binding of the mouse-derived anti-human IL-33 monoclonal antibody to human IL-33.

Figure 2 is the experimental results of the blocking of hIL-33 binding to hIL-33R by the mouse-derived anti-human IL-33 monoclonal antibody.

Fig. 3 is the experimental result of blocking the binding of hIL-33 to hIL-33R by the preferred humanized anti-human IL-33 monoclonal antibody.

Fig. 4 is the experimental result of inhibiting the secretion of IL-5 from KU812 cells by preferably humanized anti-human IL-33 monoclonal antibody.

Detailed ways

The present application provides novel anti-IL-33 antibodies or antigen-binding portions thereof that specifically bind IL-33. In a preferred embodiment, the antibody or antigen-binding portion thereof of the present application binds to human IL-33 with high affinity and inhibits the activity of IL-33. The present application also provides a polynucleotide encoding the antibody or an antigen-binding fragment thereof, a vector comprising the polynucleotide, a host cell comprising the polynucleotide or vector, a method for preparing and purifying the antibody, and the antibody Medical and biological applications of or antigen-binding fragments thereof, such as prevention or treatment of IL-33-associated diseases or disorders. The present application also encompasses methods of detecting IL-33 and modulating IL-33 activity using the antibodies or antigen-binding fragments thereof.

To facilitate understanding of this 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 disulfide bonds, as well as multimers thereof (e.g., IgM ). Each heavy chain is comprised of 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 is comprised of a light chain variable region (abbreviated VL) and a light chain constant region (abbreviated CL). The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with conserved regions called framework regions (FRs).

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 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 portions include: Fab fragments; F(ab')2 fragments; Fd fragments; Fv fragments; single chain Fv (scFv) molecules; The smallest unit of recognition composed of residues (eg, isolated CDRs). The term "antigen-binding moiety" also includes other engineered molecules such as diabodies, triabodies, tetrabodies, and minibodies, among others.

As used herein, the terms "heavy chain variable region (VH)" and "light chain variable region (VL)" refer to a single antibody variable heavy and light chain region, respectively, comprising FR1, 2, 3, and 4 and the CDRs 1, 2 and 3.

It is well known to those skilled in the art that complementarity determining regions (CDRs, usually CDR1, CDR2 and CDR3) are regions in the variable region that have the greatest influence on the affinity and specificity of antibodies. There are two common ways to define the CDR sequence of VH or VL, namely 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. USA 86:9268-9272 (1989). For the variable region sequence of a given antibody, the CDR region sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In an embodiment of the present application, Kabat is used to define CDR sequences. 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.

For the variable region sequence of a given antibody, the CDR region sequence in the variable region sequence can be analyzed in various ways, for example, it 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, for example the binding of an antibody to an antigenic epitope, for example an antibody with an affinity at least two times greater than its affinity for a non-specific antigen The ability to bind with affinity to a specific antigen. It is understood, however, that an antibody is capable of specifically binding to two or more antigens that are related in sequence. For example, antibodies of the invention can specifically bind human and non-human (e.g., non-human primate) IL-33.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of naturally occurring mutations 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 or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the heavy chain and/or the remaining part of the light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, so long as they exhibit the desired biological activity.

As used herein, the term "homology" is defined as the percentage of residues in amino acid or nucleotide sequence variants that are identical after alignment of the sequences and introduction of gaps, if desired, to achieve a maximum percentage homology. Methods and computer programs for alignment are well known in the art. "At least 80% homology" as used herein means that the homology is any value from 80% to 100%, such as 85%, 90%, 95%, 99% and the like.

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

In one aspect, the 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 chain and light chain amino acid sequences and corresponding nucleotide sequences applicable to the antibodies disclosed in the present application are exemplarily listed in the following Tables 3-7. In certain embodiments, the anti-IL-33 antibody, or antigen-binding portion thereof, comprises a HCDR3, HCDR2, or HCDR1 sequence independently selected from any of the HCDR3, HCDR2, or HCDR1 sequences shown in Table 4. In certain embodiments, the anti-IL-33 antibody of the present application may further comprise a light chain CDR independently selected from any one of the light chain CDR1, CDR2 or CDR3 sequences shown in Table 5. For example, the anti-IL-33 antibody of the present application may comprise any one of the heavy chain variable domains shown in Tables 3 and 4, optionally with the light chain variable domains shown in Tables 3 and 5 any of the pairings.

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

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

In specific embodiments, HCDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 34, 40, 46, 52, 58, 64, 70 and 76. In a preferred embodiment, HCDR2 is selected from the amino acid sequences set forth in 46, 70 and 76.

In specific embodiments, HCDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 33, 39, 45, 51, 57, 63, 69 and 75. In a preferred embodiment, HCDR1 is selected from the amino acid sequences set forth in 45, 69 and 75.

In some embodiments, an antibody heavy chain variable region disclosed herein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26 and 30. In specific embodiments, said heavy chain variable region consists of 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 amino acid sequence of the antibody heavy chain variable region disclosed herein has at least 80%, 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology. In a preferred embodiment, the heavy chain variable region has more than 99% homology with the amino acid sequence shown in SEQ ID NOs: 88, 100 or 106.

An antibody or antigen-binding portion 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 the amino acid sequences shown in SEQ ID NOs: 74 and 80, or selected from the amino acid sequences shown in SEQ ID NOs: 38, 44, 50, 56, 62 and 68 amino acid sequence. In a preferred embodiment, LCDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 50, 74 and 80.

In some embodiments, LCDR2 is selected from the amino acid sequences set forth in SEQ ID NOs: 73 and 79, or selected from the amino acid sequences set forth in SEQ ID NOs: 37, 43, 49, 55, 61 and 67. In a preferred embodiment, LCDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 49, 73 and 79.

In some embodiments, LCDR1 is selected from the amino acid sequences of SEQ ID NOs: 72 and 78, or selected from the amino acid sequences of SEQ ID NOs: 36, 42, 48, 54, 60 and 66. In a preferred embodiment, LCDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 48, 72 and 78.

In some embodiments, an antibody light chain variable region disclosed herein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28 and 32. In specific embodiments, said light chain variable region consists of an amino acid 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 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 has more than 99% homology with the sequence shown in SEQ ID NOs: 91, 103 or 109.

In some embodiments, the heavy chain or the heavy chain variable region, the light chain or the light chain variable region of the antibody disclosed herein can be substituted, deleted or added at least one on the basis of the respective specific amino acid sequences listed above Amino acid, and the obtained mutant still maintains the activity of binding IL-33.

In some embodiments, the number of amino acid substitutions, deletions or additions is 1-30, preferably 1-20, more preferably 1-10. In preferred embodiments, sequence variants differ from the original amino acid sequence by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or additions. 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 specific embodiments, the amino acid substitutions are conservative substitutions.

In preferred embodiments, the antibody disclosed herein is antibody 127H, 219H or 309H. Wherein the heavy chain sequence of the antibody 127H is shown in SEQ ID NO: 89, the light chain sequence is shown in SEQ ID NO: 92; and the heavy chain sequence of the antibody 219H is shown in SEQ ID NO: 101, the light chain sequence The sequence is shown in SEQ ID NO: 104; and the heavy chain sequence of the antibody 309H is shown in SEQ ID NO: 107, and the light chain sequence is shown in SEQ ID NO: 110.

In some embodiments, an antibody or antigen-binding portion thereof disclosed herein binds to the same epitope on interleukin-33 as antibody 20H, 127H, 177H, 219H, 309H, or competes with 20H, 127H, 177H, 219H, 309H Binds to interleukin-33.

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

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

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

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

Specifically, the inventors of the present application conducted experiments such as binding detection of anti-human IL-33 monoclonal antibody, experimental analysis of blocking the binding of IL-33 and IL-33R, and in vitro cell function detection. Experimental results show that the anti-human IL-33 monoclonal antibody disclosed herein can bind IL-33, block the signal transduction between IL-33 and IL-33R, and inhibit the occurrence of inflammatory response.

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

In some embodiments, the nucleotide molecule encoding the antibody or antigen-binding portion thereof is operably linked to a regulatory 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 can be one of pTT5, pUC57, pDR1, pcDNA3.1(+), pDHFF and pCHO 1.0. Expression vectors may include fusion DNA sequences linked with appropriate transcriptional and translational regulatory sequences.

In some embodiments, available host cells are cells containing the above-mentioned expression vectors, which may be eukaryotic cells, such as mammalian or insect host cell culture systems, which can be used for the expression of the antibody or its antigen-binding portion of the present application. For example, HEK293 cells, COS, CHO, NSO, sf9 and sf21, etc. are all applicable to the present invention. The host cell may also be a prokaryotic cell containing the above expression vector, such as DH5α, BL21(DE3) or TG1, etc.

In some embodiments, the preparation method of the anti-human IL-33 monoclonal antibody disclosed herein comprises: cultivating host cells under expression conditions, thereby expressing the anti-human IL-33 monoclonal antibody; isolating and purifying the expressed anti-human IL-33 -33 monoclonal antibody. Using the above method, the recombinant protein can be purified into a substantially uniform substance, such as a single band on SDS-PAGE electrophoresis.

In some embodiments, the anti-IL-33 antibody disclosed herein can be separated and purified by using affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high-salt buffer, changing the pH and other methods to elute the anti-IL-33 antibody bound to the affinity column.

In some embodiments, the humanized anti-human IL-33 monoclonal antibody disclosed herein is obtained by immunizing Balb/c mice with IL-33 antigen prepared in the laboratory, After the titer was high, mouse splenocytes were fused with hybridoma cells, and the hybridoma cell lines with the functional activity of inhibiting IL-33 were screened out. More specifically, through a large number of experiments, the inventors of the present application first expressed IL-33 antigen and IL-33R respectively, and on this basis, used different adjuvants to mix with IL-33 antigen to immunize mice, and then further expressed the above-mentioned small Mouse splenocytes were fused with the hybridoma cell line sp2/0, and the fused hybridoma was screened with IL-33 antigen to select a positive cell line. After verifying its ability to block the combination of IL-33 and IL-33R and indeed inhibit IL-33 After 33 functions, the target cell line was obtained. After humanizing the target molecule, the light chain and heavy chain genes were simultaneously cloned into the eukaryotic expression vector pTT5. The above expression vector was transiently transfected into HEK293 cells, cultured in serum-free medium to produce antibodies, and the humanized anti-human IL-33 monoclonal antibody was isolated or purified with Protein A affinity column.

In other embodiments, conventional techniques in the art, such as PCR mutagenesis, can be used to further alter the murine parental antibody to generate chimeric or humanized or other variant forms of the antibody. The parent antibody of the present application can be mutagenized, for example, within the complementarity determining region (CDR) domains to generate variant antibodies that can be screened for the presence of properties of interest, such as binding affinity (lower KD), IC50, specificity, preferential binding etc. Preferably, the property of interest in the variant antibody is an improvement over the property in the parental antibody. Preferably, the amino acid substitution variant antibody has at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues of the parent antibody molecule removed and a different residue inserted in its place. The sites of most interest for substitution mutagenesis are one or more CDR regions, but framework region (FR) changes are also considered. Conservative amino acid substitutions are preferred, and non-conservative amino acid changes can also be introduced and the obtained variant antibodies can be used to screen for the desired properties.

The present application also provides the use of anti-IL-33 antibody or a composition comprising anti-IL-33 antibody in the preparation of a medicament for preventing or treating IL-33-related diseases or symptoms. In some embodiments, the IL-33-related 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 can be used as anti-immune-mediated inflammation drugs. The anti-immune-mediated inflammatory response drugs referred to in this application refer to drugs that can suppress and/or treat immune-mediated inflammatory responses, for example, it can delay the development of immune-mediated inflammatory response-related symptoms and/or reduce these severity of symptoms. In some embodiments, the medicament reduces symptoms associated with an existing inflammatory response and prevents other symptoms from occurring. In some embodiments, the medicament also reduces or prevents the transfer of an inflammatory response.

In the present description and claims, the words "comprising", "comprising" and "containing" mean "including but not limited to", and are not intended to exclude other parts, additives, components, or steps.

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

Above-mentioned disclosure has described the present application generally, and following embodiment is that the present application is described further, should not be interpreted as limiting the present application. The Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, insert genes encoding proteins into vectors and plasmids, or introduce plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in numerous publications.

Example

The present disclosure is further described below in conjunction with examples, which are not intended to limit the scope of the present disclosure. The experimental method that does not indicate specific condition in the embodiment, generally according to routine condition, or according to the condition suggested by raw material or commodity manufacturer (referring to Sambrook etc., molecular cloning, laboratory handbook, Cold Spring Harbor Laboratory; Ausubel etc., contemporary molecular Biological Methods, Greene Publishing Associates, Wiley Interscience, NY). Reagents without specific sources indicated are conventional reagents purchased in the market.

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

The human IL-33 antigen sequence comes from UniProt (UniProtKB-O95760). Sangon Biotech Co., Ltd. performed codon optimization according to the codon usage preference of Cricetulus griseus, and synthesized the 112th-270th amino acid fragment, and cloned the sequence into pET-28a (+) In the vector (sourced from Sangon Biotechnology Co., Ltd.), pET-hIL-33-His was obtained. Using pET-hIL-33-His as a template, the His tag (HHHHHH) and the Flag tag (DYKDDDDK) were inserted into the N-terminal and C-terminal of the hIL-33 fragment by PCR method to obtain N-His-hIL-33, The hIL-33-Flag fragment was constructed on the pET-28a(+) expression vector, verified by sequencing, and clones with completely correct sequences were selected for prokaryotic expression production. The protein was purified from Escherichia coli supernatant by nickel column (purchased from GE) and Flag affinity chromatography (purchased from Sigma), and used for the following mouse immunization and further analysis and research.

The human IL-33R sequence comes from UniProt (UniProtKB-Q01638), and the cloning vector of this gene is purchased from Sino Biological (Cat: HG10105-M). Using the vector as a template, the hfc fragment and the Flag tag (DYKDDDDK) were inserted into the C-terminus of the extracellular segment (19-328 amino acids) of hIL-33R by PCR method to obtain hIL-33R-ECD-hfc and hIL-33R -ECD-flag fragment. The hIL-33R-ECD-hfc and hIL-33R-ECD-flag fragments were recombined and connected to the pTT5 vector, sequenced and verified, and clones with completely correct sequences were selected for eukaryotic expression and production. The hIL-33R-ECD-hfc-pTT5 and hIL-33R-ECD-flag-pTT5 vectors were transfected into HEK 293E cell line (preserved in the laboratory) by PEI method. After culturing for 5 days in Freestyle293 medium (purchased from Gibco) containing 3 mM valproic acid, the protein was purified from the cell culture supernatant using protein A (purchased from GE) and Flag affinity chromatography (purchased from Sigma) , for the following further analysis and research.

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

Example 2 Human IL-33 Antigen Immunized Mice

Dilute the hIL-33-his antigen (at a dose of 100 μg/mouse) to 75 μl with normal saline, mix it with an equal volume of Freund’s complete adjuvant, and after complete ultrasonic emulsification, treat 4-5 week-old Balb/c Mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd., animal production license number: SCXK (Shanghai) 2013-0018) were injected subcutaneously at multiple points. Three weeks later, the same antigen (at a dose of 50 μg/mouse) was also diluted to 75 μl with normal saline and mixed with an equal volume of Freund’s incomplete adjuvant. This immunization was repeated again. One week after the third immunization, the tails of all mice were cut, blood was collected to separate serum, and the serum titer was detected by ELISA coated with hIL-33-his antigen. For mice with serum antibody titers > 10000, pulse immunization was performed one week after blood collection: tail vein injection of 10 μg antigenic protein/100 μl normal saline/mouse.

The detection of titer was carried out by ELISA method: the ELISA plate was coated with hIL-33-his antigen, the coating concentration was 1 μg/ml, 100 μl per well, and coated overnight at 4°C. Plates were washed twice with PBST (PBS containing 0.5% Tween-20) and then patted dry. Add 200 μl of coating solution containing 1% BSA to each well to seal, block at room temperature for 4 hours, pat dry, and store in a -20°C refrigerator until use. During the detection, 100 μl of mouse serum of different concentrations was added to each well of the ELISA plate, and two duplicate wells were set up, and incubated at room temperature for 1.5 hours. After washing 3 times with PBST, pat dry. Add 100 μl of HRP-labeled rabbit anti-mouse Ig antibody (purchased from Sigma) diluted 1:10000 times with PBST, and incubate at room temperature for 1 hour. After washing 3 times with PBST, pat dry. Add 100 μl of chromogenic solution to each well (mix ELISA chromogenic solution A and chromogenic B solution at a volume ratio of 1:1 before use) for color development, and then add 100 μl of 2M H ₂ SO ₄ stop solution to each well to stop the reaction. Immediately measure the OD value of each well with a microplate reader (Molecular Device) at a wavelength of 450 nm.

Embodiment 3 hybridoma cell fusion and screening

Splenocytes were collected three days after the mice were immunized for fusion.

The hybridoma sp2/0 cells in good growth state (from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, the preservation number is TCM-18) were cultured in a 37°C, 5% _CO2 incubator, and the medium was changed one day before fusion. The fusion and screening process is as follows: the mouse spleen is taken, ground and washed, and then counted. Splenocytes and sp2/0 cells were mixed at a ratio of 10:1 and centrifuged at 1500 rpm for 7 minutes. Wash away the supernatant. Add 1ml of PEG (1450) within 1 minute, shake gently for 90 seconds, add 5ml of serum-free DMEM culture solution (purchased from Gibco) within 2.5 minutes, and then add 5ml of serum-free culture solution to stop the reaction, and let it stand for 5 minutes. Centrifuge at 1280 rpm for 8 minutes. According to the number of 2 million sp2/0 cells in a 96-well plate, the cells were evenly seeded into a 96-well plate, 200 μl per well. First use HAT medium containing hypoxanthine (H), methotrexate (A) and thymidine (T) to screen, change half of the medium every 3 to 4 days, and change the medium on the 10th day. Use HT medium. After 10 days, when the hybridoma cells covered more than 10% of the bottom of the 96-well plate, the supernatant was taken for ELISA detection with an enzyme label plate coated with hIL-33-his antigen. The ELISA detection method is the same as that described in Example 2. At the same time, the same ELISA detection method was used to re-screen the antibody with hIL-33-flag to eliminate the anti-His positive antibody. The hu-IL-33 positive hybridoma clones were selected and cultured in 24-well plates and subcloned by limiting dilution. After the hybridoma strain stably expressing the target antibody is obtained, the species is preserved and the library is constructed.

Example 4 Mouse-derived anti-human IL-33 monoclonal antibody blocks the binding of human IL-33 and IL-33R extracellular domain protein

The blocking of IL-33 binding to hIL-33R-ECD-hfc by murine anti-human hIL-33 monoclonal antibody was studied by ELISA method. hIL-33-his antigen coated microtiter plate, after blocking, add hIL-33R-ECD-hfc and 300 μl mouse-derived anti-human hIL-33 monoclonal antibody hybridoma cell culture supernatant in subclone, and finally add HRP Antibody for chromogenic detection. Cell lines capable of blocking hIL-33 and binding to hIL-33R-ECD-hfc were retained for the next round of subcloning.

After the preferred mouse-derived anti-human IL-33 monoclonal antibody was affinity-purified by Protein G affinity chromatography column, it was quantified by BCA method (reagents were purchased from Thermo Company). The blocking of IL-33 binding to hIL-33R-ECD-hfc by murine anti-human hIL-33 monoclonal antibody was studied by ELISA method. 307 antibodies of the established strains were analyzed, and Figure 1 shows the representative experimental results. Table 1 lists the IC ₅₀ data of some preferred antibodies, and the IC ₅₀ is all below 63ng/ml.

Table 1: Blocking of hIL-33 binding to hIL-33R-ECD-hfc by exemplary mouse-derived anti-human IL-33 monoclonal antibodies

Antibody number IC₅₀(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 5 Affinity detection of murine anti-human IL-33 monoclonal antibody binding to human IL-33

After the preferred mouse-derived anti-human IL-33 monoclonal antibody is affinity-purified through a Protein G affinity chromatography column, it is quantified by the BCA method. The EC ₅₀ of the binding of anti-human IL-33 monoclonal antibody to hIL-33-his was detected by ELISA. The detection method is as in Example 3. 1 μg/ml hIL-33-his antigen was coated on the ELISA plate, and different concentrations of mouse anti-human IL-33 monoclonal antibodies were added for detection.

307 established antibody strains were analyzed, and Figure 2 shows the representative experimental results. Table 2 lists the EC ₅₀ data of some preferred antibodies. These antibodies have high affinity to human IL-33, and the EC ₅₀ is all below 66ng/ml.

Table 2: Affinity of exemplary mouse-derived anti-human hIL-33 monoclonal antibodies to human IL-33-his

Antibody number EC₅₀(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 6 Determination of the sequence of the mouse-derived anti-human IL-33 monoclonal antibody

Trizol (purchased from Shanghai Sangon) was used to extract the total RNA of each hybridoma cell line, mRNA was reverse-transcribed into cDNA with a reverse transcription kit (purchased from Thermo Company), and Mouse Ig-Primer Set (purchased from Novagen Company) was used to Use primers to amplify the light chain variable region and heavy chain variable region genes of mouse-derived anti-human hIL-33 monoclonal antibody by PCR, then clone the PCR product into pMD18-T vector, sequence and analyze the variable region gene sequence . According to the results of various functional experiments and early druggability analysis, 8 antibodies shown in Table 3 were finally selected as lead antibodies, and the nucleotide sequences of their light and heavy chain variable regions were obtained by sequencing. The converted amino acid sequences were compared and analyzed in GenBank, and all sequences were consistent with the characteristics of mouse IgG variable region genes. According to the Kabat rule, the amino acid sequences of the light chain variable region and the heavy chain variable region of the mouse-derived anti-human IL-33 monoclonal antibody were analyzed and three CDRs were determined, as shown in Table 4 and Table 5 for details.

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

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

Table 5: Amino acid sequences of light chain CDRs of murine IL-33 antibodies

Example 7 Humanization of mouse-derived anti-human IL-33 monoclonal antibody

According to the sequence analysis results, antibodies 20, 127, 177, 219 and 309 were selected for the construction of chimeric antibodies and humanized antibodies. The amino acid sequences of the humanized antibodies are shown in Table 6 and Table 7. Construction of chimeric antibody The heavy chain variable region and light chain variable region of the murine antibody are intercepted and connected with the light and heavy chain constant regions of human IgG1 by overlapping PCR.

According to the Kabat rule, the amino acid sequences of the light chain variable region and the heavy chain variable region of the mouse-derived anti-human IL-33 monoclonal antibody were analyzed and 3 CDRs and 4 FRs were determined. Taking antibody No. 309 as an example, through homology comparison between NCBI IgBlast and human IgG germline sequence (Germline), IGHV1-46*01 was selected as the heavy chain CDR grafting template, and the mouse-derived anti-human IL-33 monoclonal The heavy chain CDR region of antibody 309 was grafted into the framework region of IGHV1-46*01 to construct a heavy chain CDR grafted antibody. Similarly, after comparing the homology with the human IgG germline sequence, IGKV1-39*01 was selected as the light chain CDR transplantation template, and the light chain CDR region of the mouse-derived anti-human IL-33 monoclonal antibody 309 was transplanted into IGKV1- The framework region of 39*01 was constructed into a light chain CDR-grafted antibody, and the resulting antibody was defined as 309-Gr (309-Grafting). At the same time, on this basis, some amino acid positions in the framework region were back-mutated. When performing back mutations, the amino acid sequence was encoded by Kabat, and the position of the site was indicated by the Kabat code. Preferably, for the heavy chain variable region, return the M at position 48 to I, the M at position 70 to L, the R at position 72 to V, and the T at position 74 to K, Revert the S at the 77th position to N, and the V at the 79th position to A; for the light chain variable region sequence, return the L at the 46th position to V, and the Y at the 49th position to S (repeated above The codes for the light chains were all based on Kabat). The above-mentioned variable region gene sequence was codon-optimized and synthesized by Sangon Biotech according to the codon usage preference of Cricetulus griseus. The synthetic humanized variable region sequence was connected with the human IgG1 constant region, and this antibody was defined as the humanized antibody of No. 309 antibody (309-Humanization, 309H).

Using the same principle as above, the remaining five antibodies were also humanized. The pTT5 vector was used to construct the transient expression vectors of the humanized heavy chain and light chain respectively, and the combination of the above-mentioned light and heavy chains was transfected transiently and expressed antibodies using the HEK293 system. HEK293 cells were cultured in Free Style 293 Expression Medium (purchased from Gibco Company) medium, and the plasmid was transferred into the cells by PEI transfection method 5 days later, the cell supernatant was collected, and the antibody was obtained after purification by Protein A.

Table 6: Nucleotide sequence and amino acid sequence of the 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 8 Humanized anti-human IL-33 monoclonal antibody blocks the binding of human IL-33 and extracellular domain protein

After the above-mentioned humanized anti-human IL-33 monoclonal antibody was affinity purified by Protein A affinity chromatography column, it was quantified by BCA method. Blocking of IL-33 binding to hIL-33R-ECD-hfc by humanized anti-human hIL-33 monoclonal antibody was studied by ELISA method. The detection method is as in Example 4. Figure 3 shows representative experimental results, and Table 8 lists the IC ₅₀ data of some antibodies. The experimental results showed that the blocking ability of the humanized anti-human IL-33 monoclonal antibody on the binding of hIL-33 to hIL-33R-ECD-hfc was significantly higher than that of the reference antibody pf158.

Table 8: Positive detection of hIL-33 binding to hIL-33R-ECD-hfc by humanized antibody pairs

Antibody number IC₅₀(ng/ml) pf158 139.60 127H 49.95 219H 93.53 309H 65.93

Example 9 Humanized anti-human IL-33 monoclonal antibody inhibits KU812 cells from secreting IL-5

KU812 cells (ATCC, CRL-2099) in good growth state were taken, counted and resuspended with recombinant hIL-33 at a final concentration of 50ng/ml to form a cell suspension of 5x10 ⁵ /ml. The culture medium is RPMI1640 medium (purchased from Gibco Company), which contains 10% fetal bovine serum (purchased from Sigma Company), 100 U/ml penicillin (purchased from Gibco Company) and 100 mg/ml streptomycin (purchased from Gibco Company) , called RPMI-1640 complete medium. After incubating at 37°C, 5% CO ₂ for 0.5 hours, different concentrations of antibodies were added. Different concentrations of humanized anti-human IL-33 monoclonal antibody (250 μg/ml, 3-fold dilution, 9 different concentrations) were diluted with culture medium solution, 100 μl per well, and added to a 96-well flat-bottomed cell culture plate ( purchased from Corning Company), and then 100 μl of cell suspension was added to each well. Two duplicate wells were set up in each group, and incubated at 37°C, 5% CO ₂ for 24 hours. The supernatant was taken to detect the secretion of IL-5 according to the instructions of the IL-5 detection kit (R&D, Cat: S5000B), and the OD value of each well was measured at a wavelength of 450 nm by a microplate reader (Molecular Device).

The above-mentioned humanized anti-human IL-33 monoclonal antibody was analyzed for the inhibitory function and activity of KU812 secreting IL-5 (Figure 4). The data of the three antibodies 127H, 219H, and 309H are shown in Table 9. The results showed that 127H and 309H had a stronger inhibitory effect on the secretion of IL-5 from KU812 cells, and their functional activity was significantly better than that of the reference antibody pf158.

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

Antibody number IC₅₀(ng/ml) pf158 658.00 127H 108.00 219H 2261.00 309H 16.45

It is to be understood that while the present disclosure has been illustrated in some form, the present disclosure is not limited to what is shown and described herein. It will be obvious to those skilled in the art that various changes may be made to the described embodiment and/or a certain feature or parameter without departing from the scope of the present application. These changes are all within the scope of the present disclosure.

Claims (12)

1. An antibody or an antigen-binding portion thereof specifically binding to interleukin-33, wherein the antibody or an 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 in SEQ ID NO:10; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12; Alternatively, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:88; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:91. 2. The antibody or antigen-binding portion thereof according to claim 1, wherein the heavy chain sequence of the antibody is shown in SEQ ID NO:89, and the light chain sequence is shown in SEQ ID NO:92. 3. The antibody or antigen-binding portion thereof according to 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 according to claim 1 or 2, wherein the antibody or antigen-binding portion thereof is capable of inhibiting the secretion of IL-5 by KU812 cells. 5. A pharmaceutical composition, characterized in that it comprises the antibody or antigen-binding portion thereof according to any one of claims 1-4 and a pharmaceutically acceptable carrier. 6. Nucleotide molecule, characterized in that it encodes the antibody or antigen-binding portion thereof according to any one of claims 1-4. 7. An expression vector, characterized in that it comprises the nucleotide molecule of claim 6. 8. The host cell, characterized in that it comprises the nucleotide molecule of claim 6 or the expression vector of claim 7. 9. A method of producing the antibody or antigen-binding portion thereof of any one of claims 1-4, wherein the method comprises: a) culturing the host cell of claim 8 under expression conditions that enable the production of the antibody or antigen-binding portion thereof, thereby expressing the antibody or antigen-binding portion thereof; and b) isolating and purifying said antibody or antigen-binding portion thereof expressed in step a). 10. Use of the antibody or antigen-binding portion thereof according to any one of claims 1-4, or the pharmaceutical composition of claim 5 in the preparation of a medicament for preventing or treating IL-33-related diseases. 11. The use according to claim 10, characterized in that 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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