CN113698484B

CN113698484B - anti-IL-23R antibodies and uses thereof

Info

Publication number: CN113698484B
Application number: CN202111193581.1A
Authority: CN
Inventors: 王晓泽; 吴振华; 徐统; 聂磊; 梅小芬; 陈刚; 王海彬; 陈旭晨; 潘晨晓
Original assignee: Haizheng Biopharmaceutical Co ltd; Zhejiang Borui Biopharmaceutical Co ltd
Current assignee: Haizheng Biopharmaceutical Co ltd; Zhejiang Borui Biopharmaceutical Co ltd
Priority date: 2021-10-13
Filing date: 2021-10-13
Publication date: 2023-07-18
Anticipated expiration: 2041-10-13
Also published as: CN113698484A; CN116948031A

Abstract

The invention relates to a novel antibody or an antibody fragment thereof of interleukin-23 receptor, which can effectively bind IL-23R, block the binding of IL-23R and human IL-23 alpha/IL-12 beta heterodimer ligand, and has good application prospect. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a VH CDR1, a VH CDR2, and a VH CDR3, and the light chain variable region comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 3. 13 or 23, and VH CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4. 14 or 24, and VH CDR3 comprises the amino acid sequence shown in SEQ NO: 5. 15 or 25; VL CDR1 comprises SEQ ID NO: 8. 18 or 28, and VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 9. 19 or 29, and VL CDR3 comprises the amino acid sequence shown in SEQ ID NO: 10. 20 or 30.

Description

anti-IL-23R antibodies and uses thereof

Technical Field

The invention belongs to the field of biological medicine, and relates to an antibody and application thereof.

Background

Interleukin 23 (IL-23) is a heterodimeric cytokine composed of two protein subunits, the p19 subunit specific for IL-23 (IL-23. Alpha.) and the p40 subunit (IL-12. Beta., p40 subunit is used in conjunction with IL-12), which are typically produced by activated macrophages or dendritic cells and act on Th17 cells expressing the IL-23 receptor to promote their proliferation and stability.

The process by which IL-23 induces IL-17 production by CD4+ T cells is mediated by activated Jak2, PI3K/Akt, STAT3 and NF-. Kappa.B. Other members of the Tyk2 and STAT families, such as STAT1, STAT4, STAT5, are also involved in this process. IL-23 receptors consist of the IL-12Rβ1 subunit and the IL-23R subunit, constituting heterodimers, in which IL-12Rβ1 binds to Tyk2 and IL-23R binds to Jak2.IL-23 binds to its receptor complex, which upon activation of Jak2 and Tyk2 downstream thereof, causes receptor complex phosphorylation and formation of STATs (1, 3,4, 5) docking sites. STATs then polymerize, phosphorylate, transfer into the nucleus and activate the corresponding genes. Only STAT4 is included in the IL-12-induced DNA-binding complex, whereas STAT3, STAT1, STAT4, and possibly STAT3/STAT4 dimer is included in the IL-23-induced DNA-binding complex. IL-23 has a strong phosphorylation of STAT3 in lymphocytes, and STAT4 has a relatively weak phosphorylation. After entering the nucleus, the phosphorylated STAT3 is combined with the promoter of the IL-17A, IL-17F gene to directly participate in the transcription and synthesis; can also be combined with a specific transcription factor RORgamma t promoter of Th17 cells to up-regulate the expression of the specific transcription factor RORgamma t promoter, thereby indirectly promoting the synthesis of IL-17A, IL-17F.

IL-23 is associated with the development of a variety of autoimmune diseases, such AS inflammatory enteritis including Crohn's Disease (CD), or Ulcerative Colitis (UC), psoriasis (PS), psoriatic Arthritis (PA), systemic Lupus Erythematosus (SLE), rheumatoid Arthritis (RA), ankylosing Spondylitis (AS).

Inflammatory Bowel Disease (IBD) is a chronic recurrent disease represented by Crohn's Disease (CD) and Ulcerative Colitis (UC), and is a idiopathic intestinal inflammatory disease affecting the ileum, rectum and colon, and is manifested clinically by diarrhea, abdominal pain, and even hematochezia. In recent years, researchers verify that IL-23 is expressed at a high level in inflammatory mucous membrane of IBD patient through immunohistochemistry and real-time quantitative PCR technology, and the high expression can promote intestinal intraepithelial lymphocyte (IEL) and NK cell activation to generate cytotoxicity, and simultaneously stimulate partial subset of T cells in IBD focus to secrete high levels of inflammatory factors such as IFN-gamma, TNF, IL-2, IL-17A and the like, thereby promoting differentiation into Th17 cells and aggravating inflammatory response.

The role of T cell abnormalities in the development and progression of psoriasis has been of increasing interest since the beginning of the 80 s of the last century. The IL-23/Th17 pathway has been more deeply known and understood in recent years. It is generally believed that dendritic cells and macrophages in the dermis of psoriatic patients produce IL-23, induce activation of Th17 cells and γδ T cells, and release inflammatory cytokines such as IL-17A, IL-17F, IL-22, IL-6 and tumor necrosis factor- α (TNF- α). IL-17A, IL-17F and IL-22 act on keratinocytes, resulting in typical pathological changes in psoriasis, such as epidermal hyperplasia, acanthosis and hyperkeratosis. Under the skin inflammation microenvironment, keratinocytes can produce more IL-23 and other inflammatory factors and chemotactic factors, so that the positive feedback circulation of IL-23/Th17 is formed, and the chronic inflammatory process of psoriasis is amplified and aggravated to chronic skin keratinization diseases.

Symptoms were found to be positively correlated with the presence of IL23R positive T lymphocytes in SLE patients, and additionally IL-23 was abnormally elevated in the blood of SLE patients compared to normal.

Rheumatoid arthritis is an autoimmune disease that is a major component of chronic inflammation of articular synovial cells. NK cells are important immune cells in the body, and can be involved in the occurrence of immune diseases and hypersensitivity reactions under specific conditions. IL-23 has close correlation with NK cell function, RA disease activity, bone destruction, etc. as an important pro-inflammatory factor.

Ankylosing Spondylitis (AS) is a chronic inflammatory disease that produces lesions primarily in the spine and sacroiliac joint. IL-23 in blood of AS patients was significantly elevated compared to normal.

The information above indicates that IL-23 mediated signaling pathways can be used as therapeutic targets for autoimmune diseases. The development of monoclonal antibodies that specifically block IL-23 signaling pathways is expected to be useful in the treatment, prevention and diagnosis of various autoimmune diseases associated with aberrant IL-23 signaling pathways.

At present, various IL-23 antibody targeting drugs are in research and development or clinical stages, wherein antibodies targeting IL-23 and IL-12 common subunit p40 are marketed as the drugs of the ustekinumab, and can simultaneously influence two signal paths of IL-23 and IL-12, so that a relatively wide biological effect can be induced, and serious side effects such as infection are possibly induced; antibodies targeting the p19 subunit specific for IL-23 are numerous, such as Guselkumab (poplar), tiltrakizumab (moesadong/suntan), risankizumab (ebuwei), and other numerous molecules under investigation in or before clinic, racetrack crowding, and severe market homogeneity.

IL-23R acts as a receptor for IL-23, and development of antibodies to the receptor is a key factor of the signal path, and has the following advantages: firstly, IL-23R is taken as a membrane protein, the expression quantity of the IL-23R is far lower than that of an IL-23 ligand, so that the effective blocking effect of the antibody can be achieved at a low dosage, and the production cost of the medicine and the economic burden of a patient can be reduced; next, IL-23R antibodies are rarely developed and represented by the molecule of the patent application by the company moesadong in 2008 (CN 101675076B), among which murine m20D7 has the best properties and is humanized as Hu20D7. However, the 20D7 molecule has not been expected to have properties for many years, and thus development of antibodies with more excellent properties is highly demanded in the art.

Disclosure of Invention

In view of the above-described needs of the prior art, the present invention provides a novel anti-IL-23R antibody, and its use in the treatment and diagnosis of diseases.

In a first aspect, the invention provides an anti-IL-23R antibody or antigen-binding fragment thereof that binds to IL-23R or a fragment thereof, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, and a light chain variable region comprising VL CDR1, VL CDR2, VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 3. 13 or 23, or consists of the amino acid sequence shown in SEQ ID NO: 4. 14 or 24, or consists of the amino acid sequence shown in SEQ NO: 5. 15 or 25 or a fragment thereof; VL CDR1 comprises SEQ ID NO: 8. 18 or 28, or consists of the amino acid sequence shown in SEQ ID NO: 9. 19 or 29, or consists of an amino acid sequence set forth in SEQ ID NO: 10. 20 or 30 or a combination thereof.

In a preferred embodiment, the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 of the heavy chain variable region of the antibody or antigen binding fragment thereof are shown in SEQ ID NOs 3,4 and 5, respectively, and the amino acid sequences of VL CDR1, VL CDR2 and VL CDR3 of the light chain variable region are shown in SEQ ID NOs 8, 9 and 10, respectively.

In a preferred embodiment, the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 of the heavy chain variable region of the antibody or antigen binding fragment thereof are shown in SEQ ID NOs 13, 14 and 15, respectively, and the amino acid sequences of VL CDR1, VL CDR2 and VL CDR3 of the light chain variable region are shown in SEQ ID NOs 18, 19 and 20, respectively; or alternatively, the process may be performed,

in a preferred embodiment, the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 of the heavy chain variable region of the antibody or antigen binding fragment thereof are shown in SEQ ID NOS 23, 24 and 25, respectively, and the amino acid sequences of VL CDR1, VL CDR2 and VL CDR3 of the light chain variable region are shown in SEQ ID NOS 28, 29 and 30, respectively.

In a further embodiment, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 1.11 or 21, or a sequence corresponding to SEQ ID NO: 1.11 or 21, which has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 6. 16 or 26, or a sequence corresponding to SEQ ID NO: 6. 16 or 26 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In some embodiments, the antibody or antigen binding fragment thereof may further comprise a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some preferred embodiments, the light chain constant region is a kappa chain constant region. In some preferred embodiments, the antibody or antigen binding fragment thereof is IgG1.

In a further embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 34. 35, 36, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31. 32, 33 or a combination thereof.

In a second aspect, there is provided a nucleic acid molecule comprising a nucleotide encoding an antibody or antigen binding fragment thereof of the invention. In some embodiments, the nucleic acid molecule encodes the heavy chain variable region and/or the light chain variable region of the antibody or antigen binding fragment thereof.

In preferred embodiments, the nucleic acid molecule encodes a heavy chain variable region having a nucleotide sequence as set forth in SEQ ID NO. 2, 12 or 22, or having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO. 2, 12 or 22. In other preferred embodiments, the nucleic acid molecule encodes a light chain variable region having a nucleotide sequence as set forth in SEQ ID NO. 7, 17 or 27, or having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO. 7, 17 or 27.

In another aspect, the invention provides a biomaterial, comprising:

(1) A vector, host cell, microorganism, or the like comprising the nucleic acid molecule of the invention; or (b)

(2) Expression products, suspensions, supernatants, etc. of the above (1).

The person skilled in the art can easily select and prepare a vector, a host cell or a microorganism comprising the coding sequence of the antibody according to the amino acid sequence of the antibody and know how to culture such a host cell or microorganism, thereby obtaining the corresponding expression product, suspension, supernatant, etc. to obtain the corresponding antibody. This is a conventional technical means in the art.

In another aspect, the invention provides a composition comprising an antibody or antigen-binding fragment thereof of the invention; preferably, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

In another aspect, there is provided a method of making an antibody or antigen-binding fragment thereof of the invention comprising: culturing the above-described host cell to express the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment from the host cell.

In another aspect, there is provided the use of an antibody of the invention or an antigen-binding fragment thereof or a nucleic acid molecule of the invention or a biological material of the invention or a composition of the invention for the preparation of a product that binds an IL-23R protein. In a preferred embodiment, the IL-23R protein is an IL-23R protein expressed on the surface of a cell membrane.

In another aspect, there is provided a method of blocking binding of IL-23R to a human IL-23 alpha/IL-12 beta heterodimer ligand comprising using an antibody or antigen-binding fragment thereof of the invention, or a composition of the invention.

In another aspect, there is provided the use of an antibody of the invention or an antigen-binding fragment thereof or a nucleic acid molecule of the invention or a biological material of the invention or a composition of the invention in the manufacture of a medicament for the treatment of a disease associated with an autoimmune disease. In a preferred embodiment, the disease is inflammatory bowel disease including Crohn's disease, or ulcerative colitis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis.

In another aspect, a pharmaceutical composition is provided comprising an antibody or antigen-binding fragment thereof of the invention and another therapeutic agent.

In another aspect, a method of administration is provided comprising administering an antibody or antigen-binding fragment thereof of the invention in combination with another therapeutic agent.

The other therapeutic agent includes, but is not limited to, other inflammation-inhibiting biomacromolecule drugs such as anti-TNFα antibodies, antibodies IL-17 antibodies, and the like.

The anti-IL-23R antibody of the invention is a blocking antibody, can block the combination of IL-23R and human IL-23 alpha/IL-12 beta heterodimer ligand, can effectively inhibit IL-23 mediated phosphorylation activation of STAT3 on HEK-Blue IL-23 cell (InvivoGen, hkb-IL 23) luciferase reporter gene cell strain, and can effectively inhibit STAT3 phosphorylation in a mouse primitive B cell strain Baf3 (Baf 3-IL23R/IL12R beta) which overexpresses IL-23R/IL12 beta 1, thereby inhibiting IL-23 mediated signal pathways. The anti-IL-23R antibody has good application potential.

Drawings

FIG. 1 shows the binding of monoclonal antibodies to IL-23R protein;

FIG. 2 shows the binding of monoclonal antibodies to IL-23R expressed by 293 cell membranes;

FIG. 3 shows the blocking of IL-23 binding to IL-23R by monoclonal antibodies mAb#2C5, mAb5E2, mAb#7A12, mAb#5D4, mAb#9A10;

FIG. 4 shows the effect of monoclonal antibodies mAb #2C5, mAb #7A12, mAb #5D4 in inhibiting IL-23 mediated activation of STAT3 phosphorylation in HEK-Blue IL-23 luciferase reporter stable cell lines;

FIG. 5 shows that monoclonal antibodies mAb #2C5, mAb #7A12, mAb #5D4 inhibit IL-23 mediated activation of signaling pathways in Baf3 stable cell lines that are overexpressed by IL-23R/IL 12. Beta.1.

Detailed Description

Definition:

in the present invention, the term "antibody" refers to an immunoglobulin capable of specifically recognizing an antigen, which encompasses a variety of antibody constructs, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, or antibody fragments.

The term "variable region" refers to the domain of an antibody heavy or light chain that recognizes and specifically binds an epitope.

CDR regions or "complementarity determining regions" refer to regions of an antibody variable region that are hypervariable in sequence and form structurally defined loops and/or contain antigen-contacting amino acid residues.

anti-IL-23R antibodies of the invention

The present invention provides anti-IL-23R antibodies having high affinity for human IL-23R protein. The antibody can effectively inhibit the combination of IL-23R and the ligand IL-23 thereof, thereby blocking the downstream signal transmission of IL-23/IL-23R, and further realizing the effect of inhibiting inflammatory reaction from the upstream.

As demonstrated in the examples, the antibodies provided herein have extremely high affinity for IL-23R and blocking effect on IL-23R/IL12R beta 1 heterodimer ligand binding to IL-23. The anti-IL-23R antibodies or antigen-binding fragments thereof of the invention comprise substitutions, insertions, or deletions. The anti-IL-23R antibodies of the invention include modifications to the light chain variable region, heavy chain variable region, light chain or heavy chain, which modifications differ in amino acid sequence from the amino acid sequence from which the antibody was derived. For example, an amino acid sequence derived from the same designated protein may be made similar to the starting sequence, e.g., have a certain percentage identity, e.g., it may be 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% identical to the starting sequence.

In certain embodiments, amino acid modifications, which may be one or more, may be introduced into the Fc region of an antibody provided herein, thereby producing an Fc variant. An Fc variant may comprise a human Fc region sequence comprising amino acid modifications at one or more amino acid positions.

"antibodies and antigen binding fragments thereof" suitable for use in the present invention include, but are not limited to, monoclonal, polyclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, deimmunized antibodies, or Fab fragments, fab 'fragments, F (ab') 2 fragments, single chain antibodies, nanobodies, and epitope-binding fragments of any of the foregoing.

In some embodiments, the antibodies of the invention may be monospecific, bispecific or multispecific. The anti-IL-23R antibody may be linked to another antibody or antibody fragment to produce a bispecific or multispecific antibody with a second or more binding specificities.

In certain embodiments, the antibodies may be further modified to add functional components, suitable moieties for antibody derivatization include, but are not limited to, the following examples: PEG, dextran, proteins, lipids, therapeutic agents or toxins. Antibodies may be modified by phosphorylation, acetylation, glycosylation, pegylation, amidation, or other protein linking, among others.

Methods of treatment and uses of antibodies

The anti-IL-23R antibodies or antigen fragments thereof provided herein can be used to diagnose, prognosticate, treat, or inhibit inflammation. The present invention relates to methods of treating an autoimmune disease in a subject by administering to a subject in need thereof an anti-IL-23R antibody or fragment thereof of the invention. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including variants and derivatives of the invention) and nucleic acids and polynucleotides encoding the antibodies of the invention (including variants and derivatives of the invention).

The anti-IL-23R antibodies of the invention may be used in combination with another therapeutic agent, including but not limited to other biomacromolecule drugs that inhibit inflammation.

The antibodies of the invention (and any additional therapeutic agents) may be administered by any suitable means, including, but not limited to, intraperitoneal, intravenous, subcutaneous, intranasal, intramuscular injection. Antibodies and variants or compositions thereof may be administered by any convenient route, for example by bolus injection or infusion, by absorption through the epithelium or skin mucosa.

anti-IL-23R antibody sequences under the present examples

Table 1: amino acid sequence numbering of VH, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, VL-CDR3 of an antibody of the invention

Table 2: antibody VH, VL DNA sequence numbering of the invention

Antibody numbering	VH sequence numbering	VL sequence numbering
			#7A12	2	7
#2C5	12	17
			#5D4	22	27

Examples

The present invention will be described below with reference to specific examples. Reagents and apparatus used in the following methods are all those commonly used in the art and are commercially available, unless explicitly stated otherwise; the methods used are all conventional in the art and can be carried out unambiguously by a person skilled in the art on the basis of the description of the examples and with corresponding results.

EXAMPLE 1 preparation of anti-IL-23 antibody murine monoclonal antibody

With a commercial human IL-23R-ECD-Fc protein (Sino Biological, cat No. H02H) 50 μg was primary immunized against 6-8 week old BALB/c mice (commercially available). The immunized mice were re-immunized with 25. Mu.g IL-23R-ECD-Fc protein on days 14 and 28 after the first immunization. The serum titers of mice after immunization were detected by enzyme-linked immunosorbent assay (Elisa). IL-23R-His (ACRObiosystem, H52H 4) antigen was diluted to 0.5. Mu.g/ml in PBS and coated on microplates overnight at4 ℃. Blocking with 1% BSA-PBS blocking solution at 37deg.C for 1 hr; after washing the plates with PBST, serum dilutions from mice were added to the plates and incubated for 1hr at 37 ℃. HRP-labeled goat anti-mouse IgG (Sigma, A0170) (1:10000) was added to the wash plate, reacted at 37℃for 0.5h, then TMB solution was added to the wash plate, reacted at room temperature in the dark for 5 minutes, and 2N H was added ₂ SO ₄ Terminating the reaction, and placing the reaction product on a microplate reader at a wavelength of 450nm to detect absorbance. On day 42 post immunization, mice of sufficient titer were boosted with 25 μg IL-23R-ECD-Fc protein and the resulting mice were used for fusion. A suspension of myeloma cells SP2/0 (ATCC) was prepared and the total SP2/0 cells were counted using a hemocytometer (Qiajin, XB-K-25). Spleen cell suspensions of the fused mice were prepared and the total cell mass was counted using a hemocytometer. Myeloma cells and spleen cells were taken according to 1:3 ratio, cell fusion was performed using an electrofusion apparatus (BTX, ECM 2001). The HAT medium was added to the fused 50ml centrifuge tube and mixed well to prepare a cell suspension. The cell suspension was then poured into a petri dish and thoroughly mixed, and the cell suspension was plated into 50 96-well plates prepared in an amount of 5 clones/well, 100. Mu.l/well, using a multichannel pipette (Thermo, F1). The cell plates after fusion were placed in a 5.5% carbon dioxide incubator (84-1A) and incubated at 37℃for 7-10 days.

anti-IL-23R antibodies in hybridoma supernatants were detected by ELISA experiments. A number of different hybridomas were identified and used for further analysis.

Example 2 detection of binding of antibodies to human IL-23R

The binding capacity of the purified antibodies to IL-23R protein was measured by the Elisa method, which is described in example 1, wherein the initial concentration of murine antibodies was 740pM and 10 gradients were obtained by 3-fold gradient dilution with PBS. Each antibody was tested in duplicate wells and EC50 was analyzed by curve fitting. As a result, high binding activity of antibodies designated mAb #5D4, mAb #5E2, mAb #7A12, mAb #2C5, mAb #9A10 to human IL-23R was confirmed (FIG. 1).

The binding of the murine mab to IL-23R on the membranes of 293 cells 293-IL23 Res (Novoprotein, XCC 05) that had been exogenously overexpressed IL-23R was analyzed by Flow Cytometry (FCM). Cells were reacted with different concentrations of mab (highest concentration 11.11nm, 3-fold gradient dilution, 9 concentration points total) at4 ℃ for 30min. After washing the cells 2 times, FITC-labeled goat anti-mouse IgG (Sigma, A0170)) was added (1:1000), and the reaction was carried out at4℃for 30 minutes in the absence of light. After washing the cells 2 times, detection was performed with a flow cytometer BD C6. Each antibody was tested in duplicate wells and EC50 was analyzed by curve fitting. The results show that the above antibodies have good binding activity to IL-23R on the membrane surface (FIG. 2).

EXAMPLE 3 blocking Effect of antibodies on IL-23R binding to IL-23 ligand

It was further examined whether these antibodies could block IL-23R binding to IL-23. IL-23R (ACROBiosystem, ILR-H5254) was diluted to 1. Mu.g/mL and coated overnight at4℃in 100. Mu.L/Kong Jiazhi ELISA plates. After washing the plate, each well was blocked with 240. Mu.L of 5% BSA-PBST at 37℃for 1h. Dilution of biotin-labeled IL-23. Alpha. With dilution (1 XPBST)&IL-12. Beta. Heterodimer protein (ACROBiosystem, ILB-H82W 6) to 0.2. Mu.g/mL; each antibody sample was diluted to 66.7nM and then subjected to 3-fold gradient dilution to obtain 10 concentration points in total. Each antibody sample was added in a gradient of 50. Mu.L and IL-23. Alpha. Each well&IL-12β50 μL,37 ℃ horizontal for 1h washing plate 3 times, HRP-streptavidin (Abcam, ab 7403) diluted into each hole added with 100 μL,37 ℃ horizontal for 1h washing 3 times. Adding 100 μL TMB color development liquid into each hole, standing at room temperature for 25min, adding 100 μL2N H ₂ SO ₄ Terminating the reaction; measuring absorbance at the wavelength of 450nm by using an enzyme-labeled instrument immediately; the data analysis is performed on the readings using analysis software. Each antibody was tested in duplicate wells and IC50 was analyzed by curve fitting. The results showed that mAb #2c5, mAb #5d4, mAb #7a12 had relatively significant blocking effect, as seen in figure 3.

Example 4 blocking IL-23 Signal pathway activation-mediated phosphorylation of STAT3 by antibodies

Based on the results of example 3, mAb #2C5, mAb #5D4, mAb #7A12 clones capable of significantly blocking IL-23R binding to IL-23 were selected for STAT3 phosphorylation assays. HEK-Blue IL-23 cells (InvivoGen, hkb-IL 23) were collected and then washed 2 times with experimental medium (DMEM, 10% heat-inactivated FBS,100U/mL penicillin, 100. Mu.g/mL streptomycin, 100. Mu.g/mL Normocin) to adjust the cell density to 5X 10 ⁵ individual/mL; dilution of HIS tagged IL-23. Alpha. With Experimental Medium&IL-12 beta heterodimer protein (ACROBiosystem, ILB-H52W 5) to 2ng/mL; mab#2c5, mab#5d4, mab#7a12 samples were diluted to 2666.67nM and subjected to 4-fold gradient dilution to obtain 9 concentration spots in total. In 96-well plates, 100. Mu.L of cell suspension was added to each well, 50. Mu.L of each gradient diluted antibody sample was placed at 37℃in 5% CO ₂ After incubation in incubator for 30min, 50. Mu.L IL-23. Alpha. Was added to each well&IL-12 beta, placed at 37 ℃, 5% CO ₂ Culturing in an incubator for 24 hours. From each test well 20. Mu. LHEK-Blue was taken ^TM IL-23 cell culture supernatant was transferred to 96-well plates with 180. Mu.L of QUANTI-Blue added to each well ^TM The solution (InvivoGen, rep-qbs) was incubated for 2h at 37 ℃. Absorbance was measured at wavelength 630nm with a microplate reader and the readings were data analyzed using analytical software, each antibody was tested in duplicate wells and IC50 was analyzed by curve fitting. The results showed that mAb #2c5, mAb #5d4, mAb #7a12 all had significant blocking effect (figure 4).

Example 5 antibodies in the overexpression of human IL-23R/IL12R beta 1 mice primary B cell strains

Inhibition of STAT5 phosphorylation in Baf3 (Baf 3-IL-23R/IL12R beta 1)

Baf3-IL-23R/IL12Rβ1 over-expression cells were constructed by lentiviral transfection. 293 cells (ATCC) in log phase, which were well conditioned, were packaged with IL-23R and IL12Rβ1 lentiviruses, respectively. First 400 million well-conditioned 293 cells were plated on a10 cm petri dish on the first day, two dishes together. The next day, plasmid containing IL-23R, pLV-EF1a-IRES-Blastcidin, which was given by the tumor medicine center of Hunan elegance Hospital, university of south China, was added to DMEM serum-free medium with a total volume of 500. Mu.L as solution A in a ratio of 1:4:3 with PxpAx2 (Addgene, # 85132) and pCMV-VSV-G (Addgene, # 8454) in a total volume of 20. Mu.g, and the mixture was shaken and then left to stand for 5 minutes. Similarly, pLV-EF1a-IRES-puro plasmid (Addgene, # 85132) carrying IL-12Rβ1 and PxpAx2 (Addgene, # 12259) and pCMV-VSV-G (Addgene, # 8454) were added in a ratio of 1:4:3 to a total of 20. Mu.g in DMEM serum-free medium having a total volume of 500. Mu.L as solution B, and mixed by shaking and allowed to stand for 5 minutes. In addition, 120. Mu.l of Lipo2000 was added to a total volume of 1ml of DMEM serum-free medium as a solution C, and the mixture was gently shaken and then allowed to stand for 5 minutes. After 5 minutes, mixing the solution A with 500 mu l C solution and the solution B with 500 mu l C solution respectively, gently shaking and mixing uniformly, standing for 20-30 minutes, and then adding into a prepared 293 cell plate respectively for virus assembly. The next day, 293 supernatant containing transfection reagent was replaced with 10ml DMEM complete medium and culture was continued, and virus-containing supernatant was collected for 72hrs after transfection for later use. When Baf3 cells (Enzymogen, CC-Y2104) are in log phase and cell density is controlled at about 40%, IL-23R lentiviral supernatant with blasticidin (Blastcidin) screening marker and IL12R 1 lentiviral supernatant with puromycin (puromycine) screening marker are simultaneously infected. After 8 hours of transfection, the medium was replaced with complete medium for cultivation. And adding 1.5 mug/ml puromycin and 15 mug/ml blasticidin 3-4 days after infection, and performing pressure screening to obtain the Baf3 with IL-23R/IL12 Rbeta 1 over-expression.

The blocking effect of candidate molecules on signal pathways was further examined using murine primordial B cells over-expressed by IL-23R/IL12rβ1. In this experiment, mab#2c5, mab#7a12, mab#5d4 were diluted 4-fold gradient with 2666.67nM as starting concentration for a total of 8 concentration points; baf3-IL-23R/IL12R beta 1 cells were collected, washed 3 times with experimental medium (1640+10% FBS) and cell density was adjusted to 5X 10 ⁶ Each well was seeded at 40. Mu.L/well in 96-well plates. Adding 20 mu L of the sample diluted in gradient into each hole, placing at 37 ℃ and 5% CO ₂ Incubate in incubator for 30min. Then, 20. Mu.L of 40ng/ml IL-23 (ACROBiosystem, ILB-H52W 5) was added to each well and the mixture was placed at 37℃in 5% CO ₂ Culturing in an incubator for 4 hours. 20. Mu.L of cell lysis mix (5X) was added per well and shaken at 300rpm for 10min at room temperature according to the instructions of the InstantOne ELISA phospho-STAT3 (Tyr 705) kit (Invitrogen, 85-86102-11). Then 50 mu L/hole cell lysate is transferred to an ELISA plate,to each test well 50. Mu.L of the detection antibody in the kit was added and the mixture was shaken at 300rpm for 1h at room temperature. 100. Mu.L of TMB color development solution was added to each measurement well 3 times with a washing solution (1X) at 200. Mu.L/Kong Xi plate, and after shaking at 300rpm at room temperature for 30min under light-shielding conditions, 100. Mu.L of stop solution was added. Absorbance was measured at wavelength 450nm with a microplate reader and the readings were data analyzed using analytical software. The results show that all three candidate molecules showed significant inhibition of activation of the signaling pathway, and the results are shown in fig. 5.

Example 6 sequencing analysis

Sequencing analysis was performed on antibodies mab#2c5, mab#7a12, mab#5d4, the sequence information obtained was as follows:

/>

sequence listing

<110> Zhejiang Borui biopharmaceutical Co., ltd

Marine biopharmaceutical Co Ltd

<120> anti-IL-23R antibodies and uses thereof

<130> DSP1F212323ZX

<140> 202111193581.1

<141> 2021-10-13

<160> 36

<170> SIPOSequenceListing 1.0

<210> 1

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Thr Thr Phe

20 25 30

Asp Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala

50 55 60

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

65 70 75 80

Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Leu Gln Gly Ser Asp Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Ile Val Ser Ser

115

<210> 2

<211> 354

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

caggttagtc tgaaagagtc tggccctgga atgttgcagc cctcccagac cctcagtctg 60

acttgttctt tttctgggtt ttcactgacc acttttgata tgggtgtagg ctggattcgt 120

cagccttcag ggaagggact ggagtggctg gcacacattt ggtgggatga tgataaatac 180

tataatccgg ccctgaagag tcggctcaca atctccaagg atacctccaa aaaccaggta 240

ttcctcaaga tcgccaatgt ggacactgca gatactgcca catactactg tgctcgatta 300

cagggttcgg actttgacta ctggggccaa ggcaccactc tcatagtctc ctca 354

<210> 3

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Thr Phe Asp Met Gly Val Gly

1 5

<210> 4

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala Leu Lys Ser

1 5 10 15

<210> 5

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Leu Gln Gly Ser Asp Phe Asp Tyr

1 5

<210> 6

<211> 115

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

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

1 5 10 15

Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr

20 25 30

Ile Glu Trp Tyr Gln Gln Gln Pro Leu Lys Ala Pro Lys Tyr Val Met

35 40 45

Glu Val Lys Lys Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Ser Phe Gly Ala Asp Arg Tyr Leu Ser Ile Ser

65 70 75 80

Asn Ile Gln Pro Glu Asp Glu Ala Ile Tyr Ile Cys Gly Val Gly Asp

85 90 95

Thr Ile Lys Glu Gln Phe Leu Tyr Val Phe Gly Gly Gly Thr Lys Val

100 105 110

Thr Val Leu

115

<210> 7

<211> 345

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

caacttgtgc tcactcaatc atcttcagcc tctttctccc tgggagcctc agcaaaactc 60

acgtgcacct tgagtagtca gcacagtacg tacaccattg aatggtatca gcaacagcca 120

ctcaaggctc ctaagtatgt gatggaggtt aagaaagatg gaagccacag tacaggtgat 180

gggattcctg atcgcttctc tggatccagc tttggtgctg atcgctacct tagcatttcc 240

aacatccagc ctgaagatga agcaatatac atctgtggtg tgggtgatac aattaaggaa 300

caatttttgt atgttttcgg cggtggaacc aaggtcactg tccta 345

<210> 8

<211> 12

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Thr Leu Ser Ser Gln His Ser Thr Tyr Thr Ile Glu

1 5 10

<210> 9

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Val Lys Lys Asp Gly Ser His Ser Thr Gly Asp

1 5 10

<210> 10

<211> 13

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Leu Tyr Val

1 5 10

<210> 11

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Thr Thr Phe

20 25 30

Asn Val Gly Ile Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala

50 55 60

Leu Lys Thr Arg Leu Ala Ile Ser Arg Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ser Arg Leu Glu Gly Asn Asn Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 12

<211> 354

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

caggttactc tgaaggagtc tggccctggg atattgcagc cctcccagac cctcagtctg 60

acttgttcct tctctgggtt ttcgcttacc acttttaatg tgggaatagg ctggattcgt 120

cagccttcag ggaagggtct ggagtggctg gcacacattt ggtgggatga tgataagtac 180

tataatccag ccctgaagac tcggctcgct atctccaggg atacctccaa aaaccaggtt 240

ttcctcaaga tcgccaatgt ggacactgca gatactgcca catactactg ttctagatta 300

gagggaaata acttcgacta ctggggccaa ggcaccactc tcacagtctc ctca 354

<210> 13

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 13

Thr Phe Asn Val Gly Ile Gly

1 5

<210> 14

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 14

His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala Leu Lys Thr

1 5 10 15

<210> 15

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 15

Leu Glu Gly Asn Asn Phe Asp Tyr

1 5

<210> 16

<211> 115

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 16

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

1 5 10 15

Ser Ala Thr Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr

20 25 30

Ile Glu Trp Tyr Gln Gln Gln Pro Leu Lys Pro Pro Lys Tyr Val Met

35 40 45

Glu Ile Lys Lys Asp Gly Ser His Asn Thr Gly Asp Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ser Ile Ser

65 70 75 80

Asn Ile Gln Pro Glu Asp Glu Ala Ile Tyr Ile Cys Gly Val Gly Asp

85 90 95

Thr Ile Thr Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Val

100 105 110

Thr Val Leu

115

<210> 17

<211> 345

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

caacttgtgc tcactcagtc atcttcagcc tctttctccc tgggagcctc agcaacactc 60

acgtgcacct tgagtagtca gcacagtaca tacaccattg aatggtatca gcaacagcca 120

ctcaagcctc ctaagtatgt gatggagatt aagaaagatg gaagccacaa cacaggtgat 180

gggattcctg atcgcttctc tggatccagc tctggtgctg atcgctacct tagcatttcc 240

aacatccagc ctgaagatga agcaatttac atctgtggtg tgggtgatac aattacggaa 300

caatttgtgt atgttttcgg cggtggaacc aaggtcactg tccta 345

<210> 18

<211> 12

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 18

Thr Leu Ser Ser Gln His Ser Thr Tyr Thr Ile Glu

1 5 10

<210> 19

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 19

Ile Lys Lys Asp Gly Ser His Asn Thr Gly Asp

1 5 10

<210> 20

<211> 13

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 20

Gly Val Gly Asp Thr Ile Thr Glu Gln Phe Val Tyr Val

1 5 10

<210> 21

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 21

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Met Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Ser Leu Tyr Ala Asn Asp Leu Leu Asp Asn Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 22

<211> 357

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

caggtccaac tgcagcagcc tggggctgaa cttgtgatgc ctggggcttc agtgaagctg 60

tcctgcaagg cttctggcta tattttctcc agttactgga tgcactgggt gaagcagagg 120

cctggacaag gccttgagtg gatcggagag attgatcctt ctgatagtta tactaactac 180

aatcaaaagt tcaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca gcagtctgac atctgaggac tctgcggtct attactgtgc aagatctctc 300

tatgctaacg acctccttga caactggggc caagggacca ctctcacagt ctcctca 357

<210> 23

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 23

Ser Tyr Trp Met His

1 5

<210> 24

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 24

Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 25

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 25

Ser Leu Tyr Ala Asn Asp Leu Leu Asp Asn

1 5 10

<210> 26

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 26

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

1 5 10 15

Asp Thr Val Ser Ile Thr Cys His Ala Ser Gln Gly Ile Thr Ser Asn

20 25 30

Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile

35 40 45

Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Asp Gln Phe Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 27

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

gacatcctga tgacccaatc tccggcctcc atgtctatac ctctgggaga cacagtcagc 60

atcacttgcc atgcaagtca gggcattacc agtaatatag ggtggttgca gcagaaacca 120

gggaaatcat ttaagggcct gatttatcat ggaaccaact tggaagatgg agttccatca 180

aggttcagtg gcagtggatc tggagcagat tattctctca ccatcagcag cctggaatct 240

gaagattttg cagactatta ctgtgtacag tatgatcagt ttccattcac gttcggctcg 300

gggacaaagc tggaaataaa a 321

<210> 28

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 28

His Ala Ser Gln Gly Ile Thr Ser Asn Ile Gly

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 29

His Gly Thr Asn Leu Glu Asp

1 5

<210> 30

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 30

Val Gln Tyr Asp Gln Phe Pro Phe Thr

1 5

<210> 31

<211> 241

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 31

Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser Gly

1 5 10 15

Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Ser Ser Ala Ser Phe Ser

20 25 30

Leu Gly Ala Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser

35 40 45

Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Leu Lys Ala Pro Lys

50 55 60

Tyr Val Met Glu Val Lys Lys Asp Gly Ser His Ser Thr Gly Asp Gly

65 70 75 80

Ile Pro Asp Arg Phe Ser Gly Ser Ser Phe Gly Ala Asp Arg Tyr Leu

85 90 95

Ser Ile Ser Asn Ile Gln Pro Glu Asp Glu Ala Ile Tyr Ile Cys Gly

100 105 110

Val Gly Asp Thr Ile Lys Glu Gln Phe Leu Tyr Val Phe Gly Gly Gly

115 120 125

Thr Lys Val Thr Val Leu Arg Ala Asp Ala Ala Pro Thr Val Ser Ile

130 135 140

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

145 150 155 160

Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys

165 170 175

Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp

180 185 190

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu

195 200 205

Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr

210 215 220

His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu

225 230 235 240

Cys

<210> 32

<211> 241

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 32

Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser Gly

1 5 10 15

Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Ser Ser Ala Ser Phe Ser

20 25 30

Leu Gly Ala Ser Ala Thr Leu Thr Cys Thr Leu Ser Ser Gln His Ser

35 40 45

Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Leu Lys Pro Pro Lys

50 55 60

Tyr Val Met Glu Ile Lys Lys Asp Gly Ser His Asn Thr Gly Asp Gly

65 70 75 80

Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu

85 90 95

Ser Ile Ser Asn Ile Gln Pro Glu Asp Glu Ala Ile Tyr Ile Cys Gly

100 105 110

Val Gly Asp Thr Ile Thr Glu Gln Phe Val Tyr Val Phe Gly Gly Gly

115 120 125

Thr Lys Val Thr Val Leu Arg Ala Asp Ala Ala Pro Thr Val Ser Ile

130 135 140

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

145 150 155 160

Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys

165 170 175

Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp

180 185 190

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu

195 200 205

Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr

210 215 220

His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu

225 230 235 240

Cys

<210> 33

<211> 233

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 33

Met Val Leu Ala Gln Phe Leu Ala Phe Leu Leu Leu Trp Phe Pro Gly

1 5 10 15

Ala Arg Cys Asp Ile Leu Met Thr Gln Ser Pro Ala Ser Met Ser Ile

20 25 30

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

35 40 45

Thr Ser Asn Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys

50 55 60

Gly Leu Ile Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser

85 90 95

Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Asp Gln

100 105 110

Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala

115 120 125

Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu

130 135 140

Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro

145 150 155 160

Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn

165 170 175

Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr

180 185 190

Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His

195 200 205

Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile

210 215 220

Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230

<210> 34

<211> 461

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 34

Met Gly Arg Leu Thr Ser Ser Phe Leu Leu Leu Ile Val Pro Ala Tyr

1 5 10 15

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

20 25 30

Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu

35 40 45

Thr Thr Phe Asp Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys

50 55 60

Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr

65 70 75 80

Asn Pro Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys

85 90 95

Asn Gln Val Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala

100 105 110

Thr Tyr Tyr Cys Ala Arg Leu Gln Gly Ser Asp Phe Asp Tyr Trp Gly

115 120 125

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

130 135 140

Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val

145 150 155 160

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro

195 200 205

Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro

210 215 220

Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly

225 230 235 240

Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile

245 250 255

Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys

260 265 270

Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln

275 280 285

Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln

290 295 300

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu

305 310 315 320

Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg

325 330 335

Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

340 345 350

Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro

355 360 365

Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr

370 375 380

Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln

385 390 395 400

Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly

405 410 415

Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu

420 425 430

Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn

435 440 445

His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455 460

<210> 35

<211> 461

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 35

Met Gly Arg Leu Thr Ser Ser Phe Leu Leu Leu Ile Val Pro Ala Tyr

1 5 10 15

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

20 25 30

Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu

35 40 45

Thr Thr Phe Asn Val Gly Ile Gly Trp Ile Arg Gln Pro Ser Gly Lys

50 55 60

Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr

65 70 75 80

Asn Pro Ala Leu Lys Thr Arg Leu Ala Ile Ser Arg Asp Thr Ser Lys

85 90 95

Asn Gln Val Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala

100 105 110

Thr Tyr Tyr Cys Ser Arg Leu Glu Gly Asn Asn Phe Asp Tyr Trp Gly

115 120 125

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

130 135 140

Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val

145 150 155 160

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro

195 200 205

Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro

210 215 220

Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly

225 230 235 240

Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile

245 250 255

Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys

260 265 270

Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln

275 280 285

Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln

290 295 300

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu

305 310 315 320

Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg

325 330 335

Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

340 345 350

Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro

355 360 365

Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr

370 375 380

Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln

385 390 395 400

Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly

405 410 415

Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu

420 425 430

Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn

435 440 445

His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455 460

<210> 36

<211> 468

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 36

Met Gly Trp Ser Cys Phe Ile Leu Phe Leu Val Ser Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Met

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe

35 40 45

Ser Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ser Leu Tyr Ala Asn Asp Leu Leu Asp Asn Trp

115 120 125

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

130 135 140

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

145 150 155 160

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

180 185 190

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

195 200 205

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

210 215 220

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

225 230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

245 250 255

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

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

305 310 315 320

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

370 375 380

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

420 425 430

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Pro Gly Lys

465

Claims

1. An anti-IL-23R antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a VH CDR1, a VH CDR2 and a VH CDR3, the light chain variable region comprises a VL CDR1, a VL CDR2, a VL CDR3,

the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 of the heavy chain variable region of the antibody or antigen binding fragment thereof are shown as SEQ ID NO. 23, 24 and 25 respectively, and the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 of the light chain variable region are shown as SEQ ID NO. 28, 29 and 30 respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO:21, or to SEQ ID NO:21 has at least 90% sequence identity; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:26, or to SEQ ID NO:26 has at least 90% sequence identity.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region hybridizes to SEQ ID NO:21 has at least 95% sequence identity; the amino acid sequence of the light chain variable region is identical to the amino acid sequence of SEQ ID NO:26 has at least 95% sequence identity.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region hybridizes to SEQ ID NO:21 has at least 96% sequence identity; the amino acid sequence of the light chain variable region is identical to the amino acid sequence of SEQ ID NO:26 has at least 96% sequence identity.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region hybridizes to SEQ ID NO:21 has at least 97% sequence identity; the amino acid sequence of the light chain variable region is identical to the amino acid sequence of SEQ ID NO:26 has at least 97% sequence identity.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region hybridizes to SEQ ID NO:21 has at least 98% sequence identity; the amino acid sequence of the light chain variable region is identical to the amino acid sequence of SEQ ID NO:26 has at least 98% sequence identity.

7. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region hybridizes to SEQ ID NO:21 has at least 99% sequence identity; the amino acid sequence of the light chain variable region is identical to the amino acid sequence of SEQ ID NO:26 has at least 99% sequence identity.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the light chain constant region is a kappa chain constant region.

10. The antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or antigen-binding fragment thereof is IgG1.

11. The antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain amino acid sequence set forth in SEQ ID NO:36, the light chain amino acid sequence is shown as SEQ ID NO: shown at 33.

12. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-11.

13. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule encodes a heavy chain variable region and a light chain variable region of the antibody or antigen binding fragment thereof.

14. The nucleic acid molecule of claim 13, wherein the nucleic acid molecule encodes a heavy chain variable region having a nucleotide sequence as set forth in SEQ ID No. 22 or having at least 90% sequence identity to SEQ ID No. 22; and, the nucleic acid molecule encodes a light chain variable region having a nucleotide sequence as set forth in SEQ ID NO. 27 or having at least 90% sequence identity to SEQ ID NO. 27.

15. The nucleic acid molecule of claim 13, wherein the nucleic acid molecule encodes a heavy chain variable region having a nucleotide sequence that has at least 95% sequence identity to SEQ ID No. 22; and, the nucleic acid molecule encodes a light chain variable region having a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO. 27.

16. The nucleic acid molecule of claim 13, wherein the nucleic acid molecule encodes a heavy chain variable region having a nucleotide sequence that has at least 96% sequence identity to SEQ ID No. 22; and, the nucleic acid molecule encodes a light chain variable region having a nucleotide sequence that has at least 96% sequence identity to SEQ ID NO. 27.

17. The nucleic acid molecule of claim 13, wherein the nucleic acid molecule encodes a heavy chain variable region having a nucleotide sequence that has at least 97% sequence identity to SEQ ID No. 22; and, the nucleic acid molecule encodes a light chain variable region having a nucleotide sequence that has at least 97% sequence identity to SEQ ID NO. 27.

18. The nucleic acid molecule of claim 13, wherein the nucleic acid molecule encodes a heavy chain variable region having a nucleotide sequence that has at least 98% sequence identity to SEQ ID No. 22; and, the nucleic acid molecule encodes a light chain variable region having a nucleotide sequence that has at least 98% sequence identity to SEQ ID NO. 27.

19. The nucleic acid molecule of claim 13, wherein the nucleic acid molecule encodes a heavy chain variable region having a nucleotide sequence that has at least 99% sequence identity to SEQ ID No. 22; and, the nucleic acid molecule encodes a light chain variable region having a nucleotide sequence that has at least 99% sequence identity to SEQ ID NO. 27.

20. A biological material, which is:

a vector, host cell or microorganism comprising the nucleic acid molecule of any one of claims 12 to 19.

21. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-11.

22. The composition of claim 21, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

23. A method of preparing the antibody or antigen-binding fragment thereof of any one of claims 1-11, comprising: culturing the host cell of claim 20 to express the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof from the host cell.

24. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or a nucleic acid molecule according to any one of claims 12 to 19 or a biological material according to claim 20 or a composition according to claim 21 or 22 for the preparation of a product that binds IL-23R protein.

25. The use of claim 24, wherein the IL-23R protein is an IL-23R protein expressed on the surface of a cell membrane.

26. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or a nucleic acid molecule according to any one of claims 12 to 19 or a biological material according to claim 20 or a composition according to claim 21 or 22 in the manufacture of a medicament for the treatment of a disease associated with autoimmune disease, inflammatory enteritis including crohn's disease or ulcerative colitis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis.

27. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-11 and another therapeutic agent that is an anti-tnfα antibody or an antibody IL-17 antibody.