CN113698484A

CN113698484A - anti-IL-23R antibodies and uses thereof

Info

Publication number: CN113698484A
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: 2021-11-26
Anticipated expiration: 2041-10-13
Also published as: CN113698484B; CN116948031A

Abstract

The invention relates to a novel antibody or an antibody fragment of interleukin-23 receptor, which can effectively bind IL-23R and 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 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 SEQ ID NO: 3. 13 or 23, VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4. 14 or 24, VH CDR3 comprises the amino acid sequence set forth in SEQ NO: 5. 15 or 25; VL CDR1 comprises SEQ ID NO: 8. 18 or 28, VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9. 19 or 29, and VL CDR3 comprises the amino acid sequence set forth 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 biomedicine, 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 (IL-23 α) and the p40 subunit (IL-12 β, p40 subunit used in conjunction with IL-12) characteristic of IL-23, and is generally produced by activated macrophages or dendritic cells and acts 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. Tyk2 and other members of the STAT family such as STAT1, STAT4, STAT5 are also involved in this process. The IL-23 receptor is composed of IL-12R β 1 subunit and IL-23R subunit, constituting a heterodimer, with IL-12R β 1 binding Tyk2 and IL-23R binding Jak 2. IL-23 binds to its receptor complex and, upon activation of Jak2 and Tyk2 downstream thereof, leads to phosphorylation of the receptor complex and formation of docking sites for STATs (1,3,4, 5). Then STATs are polymerized, phosphorylated, translocated into nucleus and activate the corresponding gene. IL-12-induced DNA-binding complexes contain STAT4 alone, while IL-23-induced DNA-binding complexes contain STAT3, STAT1, STAT4, and possibly STAT3/STAT4 dimer. In lymphocytes, IL-23 phosphorylates STAT3 more strongly and STAT4 phosphorylates less strongly. After entering the nucleus, phosphorylated STAT3 is combined with a promoter of an IL-17A, IL-17F gene and directly participates in starting transcription and synthesis; can also be combined with a specific transcription factor ROR gamma t promoter of Th17 cells to up-regulate the expression of the transcription factor ROR gamma 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 bowel disease 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 enteritis (IBD) is a chronic recurrent disease represented by Crohn's Disease (CD) and Ulcerative Colitis (UC), an idiopathic inflammatory intestinal disease affecting the ileum, rectum and colon, and is clinically manifested as diarrhea, abdominal pain and even hematochezia. In recent years, researchers verify that IL-23 is expressed at a high level in inflammatory mucous membranes of IBD patients through immunohistochemistry and real-time quantitative PCR technology, the high expression of IL-23 can promote intestinal intraepithelial lymphocytes (IEL) and NK cells to be activated to generate cytotoxicity, and meanwhile, partial T cells of IBD focus subsets are stimulated to secrete high-level inflammatory factors such as IFN-gamma, TNF, IL-2, IL-17A and the like, so that the inflammatory factors are promoted to be differentiated into Th17 cells to aggravate inflammatory reaction.

The role of T cell abnormalities in the development of psoriasis has received increasing attention since the 80 s of the last century. The IL-23/Th17 pathway has been more deeply understood and appreciated in recent years. It is believed that dendritic cells and macrophages in the dermis of patients with psoriasis produce IL-23, induce activation of Th17 cells and γ δ 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 pathological changes typical of psoriasis such as epidermal hyperplasia, acanthosis hypertrophy and hyperkeratosis. Under the microenvironment of skin inflammation, keratinocytes can produce more IL-23 and other inflammatory factors and chemokines, so that an IL-23/Th17 positive feedback cycle is formed, and the chronic inflammatory lesion process of psoriasis is amplified and aggravated to be chronic skin keratosis.

SLE patients found in symptoms and IL23R positive T lymphocytes exist in a positive correlation, in addition SLE patients in the blood IL-23 compared with normal abnormal rise.

Rheumatoid arthritis is an autoimmune disease in which chronic inflammation of joint synovial cells is predominant. In the body, NK cells are important immune cells, and can be involved in the occurrence of immune diseases and hypersensitivity under specific conditions. As an important proinflammatory factor, IL-23 has close correlation with the functions of NK cells, RA disease activity, bone destruction and the like.

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

The above information all indicate that IL-23 mediated signaling pathways can be targeted for the treatment of autoimmune diseases. Development of monoclonal antibodies that specifically block the IL-23 signaling pathway is expected to be useful in the treatment, prevention and diagnosis of various autoimmune diseases associated with abnormalities in the IL-23 signaling pathway.

At present, a plurality of targeted IL-23 antibody drugs on the market are in the development or clinical stage, wherein the antibody targeting IL-23 and IL-12 common subunit p40 is already available on the market as an ustekinumab drug, and because two signal paths of IL-23 and IL-12 can be simultaneously influenced, a wider biological effect can be caused, and more serious side effects such as infection can be possibly caused; antibodies targeting the IL-23 specific p19 subunit are numerous, such as the marketed Guselkumab (yanson), tildarkizumab (visadon/taiyang pharmaceuticals), Risankizumab (alberweib) and many other molecules in the clinic or preclinical of research, with racetrack crowding and serious market homogeneity.

IL-23R is used as a receptor of IL-23, and is a key factor of the signal path, and the development of an antibody thereof has the following advantages: firstly, the expression level of IL-23R as a membrane protein is far lower than that of an IL-23 ligand, so that the antibody can achieve an effective blocking effect at a low dose, and the production cost of the medicament and the economic burden of a patient can be reduced; secondly, the IL-23R antibody is few in research and drugs, and is represented by a molecule applied to a patent (CN 101675076B) in 2008 by Moshadong company, wherein the murine m20D7 is best in property and is named Hu20D7 through humanization. However, the 20D7 molecule has unexpected properties, and no subsequent clinical studies have been conducted for many years, so there is a strong need in the art to develop antibodies with better properties.

Disclosure of Invention

In response to 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 disease.

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

SEQ ID NOs

3,4, and 5, respectively, and the amino acid sequences of VL CDR1, VL CDR2, VL CDR3 of the light chain variable region are set forth 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 set forth in SEQ ID NOs 13, 14, and 15, respectively, and the amino acid sequences of VL CDR1, VL CDR2, VL CDR3 of the light chain variable region are set forth in SEQ ID NOs 18, 19, and 20, respectively; alternatively, the first and second electrodes may be,

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 set forth in SEQ ID NOs 23, 24, and 25, respectively, and the amino acid sequences of VL CDR1, VL CDR2, VL CDR3 of the light chain variable region are set forth in SEQ ID NOs 28, 29, and 30, respectively.

In a further embodiment, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 1.11 or 21, or a variant of SEQ ID NO: 1.11 or 21, and the light chain variable region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6. 16 or 26, or a variant of SEQ ID NO: 6. 16 or 26 have 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 IgG 1.

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 consists thereof.

In a second aspect, there is provided a nucleic acid molecule comprising nucleotides 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 present invention provides a biomaterial that is:

(1) vectors, host cells or microorganisms comprising the nucleic acid molecules of the invention, and the like; or

(2) The expression product, suspension, supernatant or the like of the above (1).

The skilled person 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 can know how to culture such host cell or microorganism to obtain the corresponding expression product, suspension, supernatant, etc. to obtain the corresponding antibody. This is all a routine technical measure 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 or antigen-binding fragment thereof of the invention or a nucleic acid molecule of the invention or a biomaterial of the invention or a composition of the invention in the manufacture of a product that binds to 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, a method of blocking IL-23R binding to a human IL-23 α/IL-12 β heterodimer ligand is provided, 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 or antigen-binding fragment thereof of the invention or a nucleic acid molecule of the invention or a biomaterial 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 combination therapy is provided comprising administering an antibody or antigen-binding fragment thereof of the invention in combination with another therapeutic agent.

Such other therapeutic agents include, but are not limited to, other inflammatory inhibiting biomacromolecule drugs such as anti-TNF α antibodies, antibody IL-17 antibodies, and the like.

The anti-IL-23R antibody is a blocking antibody, can block the binding of IL-23R and human IL-23 alpha/IL-12 beta heterodimer ligand, can effectively inhibit the phosphorylation activation of IL-23 mediated STAT3 on a luciferase reporter cell strain of HEK-Blue IL-23 cells (InvivoGen, hkb-IL23), and can effectively inhibit STAT3 phosphorylation in a mouse pro-B cell strain Baf3(Baf3-IL 23R/IL12R beta) over-expressing IL-23R/IL12 beta 1 so as to inhibit an IL-23 mediated signal pathway. The anti-IL-23R antibody of the invention has good application potential.

Drawings

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

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

FIG. 3 shows the blocking of IL-23 binding to IL-23R by monoclonal antibodies mAb #2C5, mAb5E2, mAb #7A12, mAb #5D4, mAb #9A 10;

FIG. 4 shows the effect of monoclonal antibodies mAb #2C5, mAb #7A12, mAb #5D4 in inhibition of IL-23 mediated phosphorylation activation of STAT3 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 a stable strain of Baf3 cells over-expressed IL-23R/IL12 β 1.

Detailed Description

Defining:

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

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

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 with high affinity for human IL-23R protein. The antibody can effectively inhibit the binding of IL-23R and the ligand IL-23 thereof, thereby blocking the downstream signaling of IL-23/IL-23R and realizing the effect of inhibiting the inflammatory reaction from the upstream.

As demonstrated in the examples, the antibodies provided herein have very high affinity for IL-23R and blocking of IL-23R/IL12R β 1 heterodimer ligand binding to IL-23. The anti-IL-23R antibodies or antigen-binding fragments thereof of the present invention comprise substitutions, insertions or deletions. The anti-IL-23R antibody of the invention comprises a modification of the variable region of the light chain, the variable region of the heavy chain, the light chain or the heavy chain, and the amino acid sequence of the modified antibody is different from the amino acid sequence from which the antibody is derived. For example, amino acid sequences derived from the same given protein may be made similar to the starting sequence, e.g., have a certain percent identity, e.g., it may be 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% percent 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 the antibodies provided herein, thereby generating Fc variants. An Fc variant may comprise a human Fc region sequence comprising amino acid modifications at one or more amino acid positions.

Suitable "antibodies and antigen-binding fragments thereof" for use in the present invention include, but are not limited to, monoclonal, polyclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, de-immunogenic 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 can 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 portions for antibody derivatization include, but are not limited to, the following examples: PEG, dextran, proteins, lipids, therapeutic agents or toxins. Antibodies can be modified by phosphorylation, acetylation, glycosylation, pegylation, amidation, or other protein linkages, among others.

Antibody treatment methods and uses

The anti-IL-23R antibodies or antigenic fragments thereof provided by the invention can be used for diagnosis, prognosis, treatment or inhibition of inflammation. The present invention relates to methods of treating an autoinflammatory disease in a subject by administering an anti-IL-23R antibody or fragment thereof of the invention to a subject in need thereof. 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 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. The antibody and variants or compositions thereof may be administered by any convenient route, for example by bolus injection or infusion, absorption through the epithelium or skin mucosa.

anti-IL-23R antibody sequences exemplified herein

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: DNA sequence numbering of the antibodies VH, VL of the invention

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

Examples

The present invention will now be described with reference to specific examples. Unless otherwise specified, the reagents and apparatus used in the following methods are those commonly used in the art and are commercially available; the methods used are conventional in the art and can be carried out unambiguously by the person skilled in the art from the description of the examples and corresponding results are obtained.

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

Primary immunization of 6-8 week old BALB/c mice (commercially available) was performed with 50. mu.g of commercial human IL-23R-ECD-Fc protein (Sino Biological, cat H02H). On days 14 and 28 after the first immunization, the immunized mice were re-immunized with 25. mu.g of IL-23R-ECD-Fc protein. The serum titer of the immunized mice is detected by an enzyme-linked immunosorbent assay (Elisa) method. IL-23R-His (ACROBIOSystem, H52H4) antigen was diluted to 0.5. mu.g/ml in PBS and the plates were coated overnight at4 ℃. Then blocking for 1h at 37 ℃ by using 1% BSA-PBS blocking solution; after PBST washing of the plates, serum dilutions from mice were added to the plates and incubated for 1hr at 37 ℃. Adding goat anti-mouse IgG (Sigma, A0170) (1: 10000) marked by HRP into the washing plate, reacting for 0.5h at 37 ℃, then adding TMB solution into the washing plate, reacting for 5 minutes at room temperature in a dark place, adding 2N H₂SO₄The reaction is stopped, and the reaction solution is placed on a microplate reader for detecting the absorbance at the wavelength of 450 nm. On day 42 post-immunization, mice with sufficient titer were boosted with 25 μ g of 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 amount of SP2/0 cells was calculated using a hemocytometer (Qiaking, XB-K-25). Spleen cell suspensions of the fusion mice were prepared and counted using a hemocytometer to calculate the total cell mass. Myeloma and splenocytes were taken as 1: 3 ratio ofFor example, cell fusion was carried out using an electrofusion apparatus (BTX, ECM 2001). Adding HAT culture medium into the fused 50ml centrifuge tube, and mixing uniformly to prepare cell suspension. The cell suspension was then poured into a petri dish and mixed well, and the cell suspension was plated into prepared 50 96-well plates in an amount of 5 clones/well, 100. mu.l/well, using a multi-channel pipette (Thermo, F1). The fused cell plate was placed in a 5.5% carbon dioxide incubator (84-1A) and incubated at a constant temperature of 37 ℃ for 7-10 days.

anti-IL-23R antibody was detected in hybridoma supernatants using an ELISA assay. 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 antibody to the IL-23R protein was determined by the Elisa method as described in example 1, wherein the initial concentration of the murine antibody was 740pM and a total of 10 gradients were obtained by 3-fold gradient dilution with PBS. Each antibody was tested in duplicate and EC50 was analyzed by curve fitting. As a result, high binding activity of the antibodies designated mAb #5D4, mAb #5E2, mAb #7A12, mAb #2C5, mAb #9A10 to human IL-23R was confirmed (FIG. 1).

The above murine mAbs were analyzed by Flow Cytometry (FCM) for binding to IL-23R on membranes of 293 cells 293-IL23 Res (Novoprotein, XCC05) which exogenously over-expressed IL-23R. Cells were reacted with different concentrations of mAb (maximum concentration of 11.11nM, 3-fold gradient dilution, total of 9 concentration points) at4 ℃ for 30 min. After washing the cells 2 times, FITC-labeled goat anti-mouse IgG (Sigma, A0170)) (1:1000) was added and the reaction was carried out at4 ℃ for 30 minutes in the absence of light. After washing the cells 2 times, detection was performed using a flow cytometer BD C6. Each antibody was tested in duplicate and EC50 was analyzed by curve fitting. The results showed that the above antibodies all had good binding activity to IL-23R on the membrane surface (FIG. 2).

Example 3 blocking of IL-23R binding to IL-23 ligand by antibodies

Further testing whether these antibodies can block the binding of IL-23R to IL-23. IL-23R (ACROBIOSystem, ILR-H5254) was diluted to 1. mu.g/mL, added to the microplate at 100. mu.L/well, and coated overnight at4 ℃. After washing the plate, 240 mu L of 5 percent is added into each holeBSA-PBST blocking at 37 ℃ for 1 h. Dilution of Biotin-labeled IL-23. alpha. with Diluent (1 XPBST)&IL-12 β heterodimer protein (ACROBIOSystem, ILB-H82W6) to 0.2 μ g/mL; each antibody sample was diluted to 66.7nM followed by a 3-fold gradient dilution, yielding a total of 10 concentration points. Each well was loaded with 50. mu.L of each antibody sample diluted in a gradient and IL-23. alpha&IL-12. beta.50. mu.L, horizontal at 37 ℃ 1h plate washing 3 times, HRP-streptavidin (Abcam, Ab7403) diluted and added to each well 100. mu.L, horizontal at 37 ℃ 1h plate washing 3 times. Adding 100 μ L of TMB color developing solution into each well, standing horizontally at room temperature in dark place for 25min, and adding 100 μ L of 2N H₂SO₄Terminating the reaction; immediately measuring absorbance at the wavelength of 450nm by using an enzyme-labeling instrument; data analysis is performed on the readings using analysis software. Each antibody was tested in duplicate and IC50 was analyzed by curve fitting. The results show that mAb #2C5, mAb #5D4, mAb #7a12 have relatively significant blocking effect, and the results are shown in fig. 3.

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

Based on the results of example 3, mAb #2C5, mAb #5D4, mAb #7A12 clones that significantly blocked IL-23R binding to IL-23 were selected for STAT3 phosphorylation assays. HEK-Blue IL-23 cells (InvivoGen, hkb-IL23) were collected and washed 2 times with experimental media (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 10⁵Per mL; dilution of HIS-tagged IL-23 alpha with Experimental Medium&IL-12 β heterodimer protein (ACROBIOSystem, ILB-H52W5) to 2 ng/mL; mAb #2C5, mAb #5D4, mAb #7a12 samples were diluted to 2666.67nM and further diluted with a 4-fold gradient to obtain 9 concentration points in total. In a 96-well plate, 100. mu.L of cell suspension and 50. mu.L of each antibody sample diluted in a gradient were added to each well, and the mixture was incubated at 37 ℃ and 5% CO₂After incubation in an incubator for 30min 50. mu.L of IL-23. alpha. was added per well&IL-12 beta, incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 24 h. Remove 20 μ LHEK-Blue from each test well^TMIL-23 cell culture supernatants were transferred to 96-well plates and 180. mu.L QUANTI-Blue was added to each well^TMThe solution (InvivoGen, rep-qbs) was incubated at 37 ℃ for 2 h. Measuring absorbance at a wavelength of 630nm with a microplate reader andthe readings were subjected to data analysis using analytical software to run each antibody in duplicate and the IC50 was analyzed by curve fitting. The results showed that mAb #2C5, mAb #5D4, and mAb #7a12 all had significant blocking effects (fig. 4).

EXAMPLE 5 antibody in mouse ProB cell line overexpressing human IL-23R/IL12R beta 1

Inhibition of STAT5 phosphorylation in Baf3(Baf3-IL-23R/IL 12R. beta.1)

The transfection method of lentivirus is adopted to construct Baf3-IL-23R/IL12R beta 1 overexpression cells. Good, at log phase 293 cells (ATCC) were taken for IL-23R and IL12R β 1 lentiviral packaging, respectively. First, 400 million well-conditioned 293 cells were plated in 10 cm culture dishes on the first day, and then two dishes were plated. The plasmid of pLV-EF1a-IRES-Blastcidin (the plasmid was given by the tumor medicine center of Xiangya hospital, university of Central south China) carrying IL-23R was added to a serum-free DMEM medium containing PxpAx2(Addgene, #85132) and pCMV-VSV-G (Addgene, #8454) in a ratio of 1:4:3 in a total amount of 20. mu.g as solution A in a total volume of 500. mu.L, mixed by shaking and allowed to stand for 5 minutes the next day. Similarly, pLV-EF1a-IRES-puro plasmid (Addgene, #85132) carrying IL-12R β 1 and PxpAx2(Addgene, #12259) and pCMV-VSV-G (Addgene, #8454) were added to a serum-free DMEM medium in a total volume of 500. mu.L at a ratio of 1:4:3 in a total amount of 20. mu.g, and allowed to stand for 5 minutes with shaking as solution B. In addition, 120. mu.l of Lipo2000 was added to a total volume of 1ml of DMEM serum-free medium as a C solution, gently shaken and mixed, and then allowed to stand for 5 minutes. After 5 minutes, respectively mixing the solution A with the solution 500 mu l C and the solution B with the solution 500 mu l C, gently shaking and uniformly mixing, standing for 20-30 minutes, and respectively adding into prepared 293 cell plates for virus assembly. The next day, 293 supernatants containing transfection reagents were replaced with 10ml DMEM complete medium and incubation continued, and virus-containing supernatants were collected 72hrs post transfection for use. While Baf3 cells (enzyme research organism, CC-Y2104) were in log phase and cell density was controlled at around 40% while infecting IL-23R lentiviral supernatant with blasticidin (Blastcidin) selection marker and IL12R β 1 lentiviral supernatant with puromycin (puromycin) selection marker. After 8 hours of transfection, the medium was replaced with complete medium for culturing. After infection for 3-4 days, 1.5. mu.g/ml puromycin and 15. mu.g/ml blasticidin are added for pressure screening to obtain Baf3 with over-expression of IL-23R/IL12R beta 1.

The blocking effect of the candidate molecules on the signal path is further tested by using IL-23R/IL12R beta 1 over-expressed mouse pro-B cells. In this experiment, mAb #2C5, mAb #7a12, mAb #5D4 were diluted 4-fold in a gradient starting at 2666.67nM for 8 concentration points; baf3-IL-23R/IL12R beta 1 cells were harvested, washed 3 times with experimental medium (1640+ 10% FBS), and cell density adjusted to 5X 10⁶one/mL, 40. mu.L/well was seeded in 96-well plates. 20 μ L of the sample diluted in gradient was added to each well, and the mixture was incubated at 37 ℃ with 5% CO₂Incubate in incubator for 30 min. Then 20. mu.L of 40ng/ml IL-23(ACROBIOSystem, ILB-H52W5) was added to each well and the mixture was incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 4 h. mu.L of cell lysis mix (5X) was added per well followed by Instantone ELISA phosphor-STAT 3(Tyr705) kit (Invitrogen, 85-86102-11) for 10min at 300 rpm. Then 50. mu.L/well of cell lysate was transferred to an ELISA plate, 50. mu.L of detection antibody in the kit was added to each test well, and the mixture was shaken at room temperature and 300rpm for 1 hour. After washing the plate 3 times with a washing solution (1X) at 200. mu.L/well, 100. mu.L of TMB developing solution was added to each measurement well, and 100. mu.L of stop solution was added thereto after shaking at 300rpm in the dark at room temperature for 30 min. Absorbance was measured at a wavelength of 450nm with a microplate reader and data analysis was performed on the readings using analysis software. The results show that all three candidate molecules exhibit significant inhibition of signal pathway activation, and the results are shown in fig. 5.

Example 6 sequencing analysis

Sequencing analysis was performed on the antibodies mAb #2C5, mAb #7A12, mAb #5D4, and the sequence information obtained was as follows:

Claims

1. an anti-IL-23R 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 SEQ ID NO: 3. 13 or 23, and VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4. 14 or 24, VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ NO: 5. 15 or 25 or consists thereof; VL CDR1 comprises SEQ ID NO: 8. 18 or 28, and VL CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9. 19 or 29, and VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10. 20 or 30 or consists thereof;

preferably, 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, VL CDR3 of the light chain variable region are shown in SEQ ID NOs 8, 9, and 10, respectively; alternatively, the first and second electrodes may be,

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 respectively shown in SEQ ID NO 13, 14 and 15, and the amino acid sequences of VL CDR1, VL CDR2 and VL CDR3 of the light chain variable region are respectively shown in SEQ ID NO 18, 19 and 20; alternatively, the first and second electrodes may be,

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 NO 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 NO 28, 29 and 30 respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 1.11 or 21, or a variant of SEQ ID NO: 1.11 or 21 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 shown as SEQ ID NO: 6. 16 or 26, or a variant of SEQ ID NO: 6. 16 or 26 have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, 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; preferably, the light chain constant region is a kappa chain constant region; further preferably, the antibody or antigen binding fragment thereof is IgG 1; preferably, 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 consists thereof.

4. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 3; preferably, 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; further preferably, 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 with SEQ ID NO 2, 12 or 22; and/or 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 with SEQ ID NO 7, 17 or 27.

5. A biomaterial, being:

(1) a vector, a host cell, a microorganism or the like comprising the nucleic acid molecule of claim 4; or

(2) Expression product, suspension, supernatant of the above (1).

6. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3; preferably, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

7. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising: culturing the host cell of claim 5 to express the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof from the host cell.

8. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or a nucleic acid molecule according to claim 4 or a biological material according to claim 5 or a composition according to claim 6 for the preparation of a product that binds to an IL-23R protein; preferably, the IL-23R protein is an IL-23R protein expressed on the surface of a cell membrane.

9. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or a nucleic acid molecule according to claim 4 or a biological material according to claim 5 or a composition according to claim 6 in the manufacture of a medicament for the treatment of a disease associated with an autoimmune disease; preferably, the disease associated with autoimmune disease is inflammatory bowel disease including crohn's disease, or ulcerative colitis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 3 and another therapeutic agent; preferably, the additional therapeutic agent includes, but is not limited to, other inflammatory inhibiting biomacromolecule drugs, such as anti-TNF α antibodies or antibody IL-17 antibodies.