Anti-human interleukin 23 monoclonal antibody and application thereof

Abstract

The present invention relates to an anti-human interleukin 23 (hIL-23) monoclonal antibody and an application thereof. The anti-human interleukin 23 (hIL-23) monoclonal antibody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein (a): the amino acid sequence of the CDR-H1 is as shown in SEQ ID NO: 1, (b): the amino acid sequence of the CDR-H2 is as shown in SEQ ID NO: 2, (c): the amino acid sequence of the CDR-H3 is as shown in SEQ ID NO: 3, (d): the amino acid sequence of the CDR-L1 is as shown in SEQ ID NO: 4, (e): the amino acid sequence of the CDR-L2 is as shown in SEQ ID NO: 5, and (f): the amino acid sequence of the CDR-L3 is as shown in SEQ ID NO: 6. Compared with existing anti-human interleukin 23 monoclonal antibodies (Guselkumab and Risankizumab), the hIL-23 binding affinity of the anti-human interleukin 23 (hIL-23) monoclonal antibody is equivalent, but the antagonistic activity at the cellular level is superior to Guselkumab, and equivalent to Risankizumab.

Description

Anti-human interleukin-23 monoclonal antibodies and their applications Technical Field

[0001] This invention relates to the field of antibody drugs. Specifically, this invention relates to monoclonal antibodies against human interleukin-23 (hIL-23) and their applications. Background Technology

[0002] IL-23 is primarily produced by activated dendritic cells, macrophages, and monocytes. It is a member of the IL-12 heterodimer cytokine family and is mainly composed of two subunits: IL-23p19 and IL-12 / IL-23p40. The IL-23 receptor includes two subunits: IL-12 receptor β1 and IL-23 receptor. IL-23 exerts its biological functions by interacting with the receptors IL-23R and IL-12Rβ1 expressed on the surface of T cells, NK cells, and monocytes / macrophages / dendritic cells, activating downstream signaling pathways. IL-23 primarily acts on Th17 cells, inducing the production of pro-inflammatory cytokines such as IL-17A, IL-17F, IL-21, and IL-22, playing a crucial role in autoimmune and inflammatory diseases such as psoriasis, psoriatic arthritis, multiple sclerosis, Crohn's disease, and inflammatory bowel disease.

[0003] IL-23-mediated signal transduction and biological effects are associated with a variety of diseases, including rheumatoid arthritis, juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, gastric ulcer, inflammatory bowel disease, ulcerative colitis, acute pancreatitis, primary cholecystitis, Hashimoto's thyroiditis, systemic lupus erythematosus, iridocyclitis, uveitis, optic neuritis, idiopathic pulmonary fibrosis, and systemic vasculitis. Wegener's granulomatosis, allergic / atopic diseases, asthma, allergic rhinitis, eczema, adult respiratory distress syndrome, allergic contact dermatitis, vitiligo, psoriasis, alopecia areata, pemphigus, scleroderma, allergic / atopic conjunctivitis, allergic pneumonia, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, Graves' disease, Raynaud's disease, type B insulin-resistant diabetes, myasthenia gravis, nephrotic syndrome, nephritis, glomerulonephritis, acute renal failure, etc.

[0004] Johnson & Johnson and AbbVie's monoclonal antibody drugs targeting IL-23, Guselkumab (trade name Tremfya) and Risankizumab (trade name SKYRIZI), have been approved by the U.S. FDA for the treatment of psoriasis and psoriatic arthritis, and are also undergoing Phase III clinical trials for Crohn's disease and inflammatory bowel disease.

[0005] Summary of the Invention

[0006] The purpose of this invention is to provide a novel anti-human interleukin-23 (hIL-23) monoclonal antibody, a pharmaceutical composition comprising the monoclonal antibody, and the pharmaceutical use of the monoclonal antibody.

[0007] That is, the present invention includes:

[0008] 1. An isolated anti-human interleukin-23 monoclonal antibody comprising three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein:

[0009] (a) The amino acid sequence of CDR-H1 (in this specification, CDR-H1 represents heavy chain CDR1) is shown in SEQ ID NO: 1 (NHEMS);

[0010] (b) The amino acid sequence of CDR-H2 (in this specification, CDR-H2 represents heavy chain CDR2) is shown in SEQ ID NO: 2 (IITTSDTTYYATWAKG);

[0011] (c) The amino acid sequence of CDR-H3 (in this specification, CDR-H3 represents heavy chain CDR3) is shown in SEQ ID NO: 3 (VDIVLLSVTSRI);

[0012] (d) The amino acid sequence of CDR-L1 (in this specification, CDR-L1 represents light chain CDR1) is shown in SEQ ID NO: 4 (QASQSVSTYLS);

[0013] (e) The amino acid sequence of CDR-L2 (in this specification, CDR-L2 represents the light chain CDR2) is shown in SEQ ID NO: 5 (GASNLES); and

[0014] (f) The amino acid sequence of CDR-L3 (in this specification, CDR-L3 represents light chain CDR3) is shown in SEQ ID NO: 6 (QSGYVFAGLT).

[0015] 2. The monoclonal antibody according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein,

[0016] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and its amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGFSLSNHEMSWVRQAPGKGLEWIGIITTSDTTYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVDIVLLSVTSRIWGQGTLVTVSS; and,

[0017] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, and its amino acid sequence is DVVMTQSPSSLSASVGDRVTITCQASQSVSTYLSWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSGYVFAGLTFGGGTKVEIK.

[0018] 3. An isolated nucleic acid encoding any of the aforementioned monoclonal antibodies.

[0019] 4. A host cell comprising the nucleic acid as described in item 3.

[0020] The nucleic acid can be present on a vector. The vector can be of any type, for example, a recombinant vector such as an expression vector. Any of a variety of host cells can be used. In one embodiment, the host cell is a prokaryotic cell, for example, *Escherichia coli* (E. coli). In another embodiment, the host cell is a eukaryotic cell, for example, a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

[0021] 5. A method for producing a monoclonal antibody, the method comprising culturing a host cell according to claim 4 to produce any of the aforementioned monoclonal antibodies.

[0022] The method includes producing the monoclonal antibody by expressing a recombinant vector encoding the anti-human interleukin-23 monoclonal antibody in suitable host cells. In some embodiments, the method includes culturing host cells containing nucleic acids encoding the anti-human interleukin-23 monoclonal antibody to express the nucleic acids. The method may further include recovering the anti-human interleukin-23 monoclonal antibody from a host cell culture or host cell culture medium.

[0023] 6. A pharmaceutical composition comprising any of the aforementioned monoclonal antibodies and a pharmaceutically acceptable carrier.

[0024] The pharmaceutical composition may further comprise additional therapeutic agents (e.g., different anti-human interleukin-23 (hIL-23) antibodies).

[0025] 7. The pharmaceutical composition according to item 6, for treating diseases related to IL-23-mediated signal transduction.

[0026] 8. The pharmaceutical composition according to claim 7, wherein the diseases associated with the IL-23-mediated signal transduction are rheumatoid arthritis, juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, gastric ulcer, inflammatory bowel disease, ulcerative colitis, acute pancreatitis, primary biliary cirrhosis, Hashimoto's thyroiditis, systemic lupus erythematosus, iridocyclitis, uveitis, optic neuritis, idiopathic pulmonary fibrosis, and systemic lupus erythematosus. Systemic vasculitis / Wegener's granulomatosis, allergic / atopic diseases, asthma, allergic rhinitis, eczema, adult respiratory distress syndrome, allergic contact dermatitis, vitiligo, psoriasis, alopecia areata, pemphigus, scleroderma, allergic conjunctivitis, allergic pneumonia, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, Graves' disease, Raynaud's disease, type B insulin-resistant diabetes, myasthenia gravis, nephrotic syndrome, nephritis, glomerulonephritis and / or acute renal failure.

[0027] 9. Use of any of the aforementioned monoclonal antibodies in the preparation of medicaments for the treatment of diseases related to IL-23-mediated signal transduction.

[0028] 10. The use according to item 9, wherein the diseases associated with the IL-23-mediated signal transduction are rheumatoid arthritis, juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, gastric ulcer, inflammatory bowel disease, ulcerative colitis, acute pancreatitis, primary biliary cirrhosis, Hashimoto's thyroiditis, systemic lupus erythematosus, iridocyclitis, uveitis, optic neuritis, idiopathic pulmonary fibrosis, and systemic... Vasculitis / Wegener's granulomatosis, allergic / atopic diseases, asthma, allergic rhinitis, eczema, adult respiratory distress syndrome, allergic contact dermatitis, vitiligo, psoriasis, alopecia areata, pemphigus, scleroderma, allergic conjunctivitis, allergic pneumonia, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, Graves' disease, Raynaud's disease, type B insulin-resistant diabetes, myasthenia gravis, nephrotic syndrome, nephritis, glomerulonephritis and / or acute renal failure.

[0029] 11. A method for treating diseases related to IL-23-mediated signal transduction, comprising:

[0030] Administer to the subject who requires the use of the monoclonal antibody or the pharmaceutical composition described in any of the preceding claims.

[0031] 12. The method according to claim 11, wherein the diseases associated with the IL-23-mediated signal transduction are rheumatoid arthritis, juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, gastric ulcer, inflammatory bowel disease, ulcerative colitis, acute pancreatitis, primary biliary cirrhosis, Hashimoto's thyroiditis, systemic lupus erythematosus, iridocyclitis, uveitis, optic neuritis, idiopathic pulmonary fibrosis, and systemic lupus erythematosus. Systemic vasculitis / Wegener's granulomatosis, allergic / specific diseases, asthma, allergic rhinitis, eczema, adult respiratory distress syndrome, allergic contact dermatitis, vitiligo, psoriasis, alopecia areata, pemphigus, scleroderma, allergic conjunctivitis, allergic pneumonia, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, Graves' disease, Raynaud's disease, type B insulin-resistant diabetes, myasthenia gravis, nephrotic syndrome, nephritis, glomerulonephritis and / or acute renal failure.

[0032] 13. The method according to item 12, wherein the systemic vasculitis is Wegener's granulomatosis.

[0033] The effects of the invention

[0034] This invention provides a novel anti-human interleukin-23 (hIL-23) monoclonal antibody that has comparable affinity for hIL-23 to existing anti-human interleukin-23 monoclonal antibodies (Guselkumab and Risankizumab), but exhibits superior antagonistic activity at the cellular level compared to Guselkumab, while being comparable to Risankizumab.

[0035] It should be noted that Risankizumab It has been approved for marketing in Japan, the United States, and the European Union, and clinical trial results show that it is more effective than Johnson & Johnson's blockbuster anti-inflammatory drug in treating moderate to severe plaque psoriasis. (ustekinumab) and AbbVie's best-selling anti-inflammatory drug (adalimumab).

[0036] The monoclonal antibody of this invention exhibits antagonistic activity comparable to that of Risankizumab at the cellular level, and is expected to demonstrate good clinical efficacy in the prevention and treatment of related diseases. Attached Figure Description

[0037] Figure 1 shows the electrophoresis results of the constructed QX004N(HZD90-32) transient transfection expression plasmid. In the figure, M: Marker; Band 1: PCR product 90VH-Hu18; Band 2: pHZDCH, HindIII / NheI; Band 3: PCR product 90VK-Hu9; Band 4: pHZDCK, HindIII / BsiWI.

[0038] Figure 2 is a flowchart of the instantaneous transition expression.

[0039] Figure 3 shows the electrophoretic detection results of QX004N(HZD90-32).

[0040] Figure 4 shows the inhibition of IL-23-induced HEK Blue by QX004N (HZD90-32), gusselukab, and risankizumab. TM STAT3 phosphorylation activity in IL-23 cells.

[0041] Figure 5 shows the inhibition of IL-17A release from mouse spleen cells by QX004N (HZD90-32), Guselkumab, and Risankizumab.

[0042] Figure 6 shows the inhibition of IL-23-induced IFN-γ release from human NK cells by QX004N (HZD90-32), Guselkumab, and Risankizumab.

[0043] Figure 7 shows the specificity of QX004N (HZD90-32), Guselkumab, and Risankizumab binding to the IL-23 p19 subunit. Detailed Implementation

[0044] The technical terms mentioned in this specification have the same meanings as those commonly understood by those skilled in the art, and in case of any conflict, the definitions in this specification shall prevail.

[0045] Generally speaking, the terms used in this specification have the following meanings.

[0046] In this specification, an “isolated” antibody is an antibody that has been separated from components of its native environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, which is determined by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0047] In this specification, "monoclonal antibody" means an antibody derived from a substantially homologous group of antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, and such variants are typically present in trace amounts, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody articles). Unlike polyclonal antibody articles, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody article targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody to be used according to the invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0048] In this specification, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K0). D Affinity can be measured using common methods known in the art.

[0049] In this specification, human interleukin 23 (hIL-23) refers to a human-derived protein, a heterodimer composed of two subunits, p19 and p40. The amino acid sequence of p19 is shown in SEQ ID NO: 9, and the amino acid sequence of p40 is shown in SEQ ID NO: 10, wherein the underlined portion represents the signal peptide.

[0050] SEQ ID NO: 9:

[0051]

[0052] SEQ ID NO: 10:

[0053]

[0054] In this specification, "anti-human interleukin-23 monoclonal antibody" means a monoclonal antibody that can bind to human interleukin-23 with sufficient affinity, such that the monoclonal antibody can be used as a diagnostic and / or therapeutic agent targeting human interleukin-23.

[0055] The anti-human interleukin-23 (IL-23) monoclonal antibody of the present invention does not bind to proteins unrelated to the target. Here, "unrelated protein" refers to proteins other than human interleukin-23, which is the target; and "does not bind" means that, when the binding ability of the anti-human interleukin-23 (IL-23) monoclonal antibody of the present invention to human interleukin-23, which is its target, is taken as 100%, the binding ability of the anti-human interleukin-23 (IL-23) monoclonal antibody of the present invention to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0056] The anti-human interleukin-23 (IL-23) monoclonal antibody of the present invention may not bind to interleukin-23 from other animal species. Here, "other animal species" refers to animal species other than humans, such as rhesus monkeys, cynomolgus monkeys, rats, mice, etc.; here, "not binding" means that when the binding ability of the anti-human interleukin-23 (IL-23) monoclonal antibody of the present invention to human interleukin-23 as its target is taken as 100%, the binding ability of the anti-human interleukin-23 (IL-23) monoclonal antibody of the present invention to interleukin-23 from other animal species is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0057] The anti-human interleukin-23 (IL-23) monoclonal antibody of the present invention has equilibrium dissociation constants (K0) of ≤1 μM, ≤100 nM, ≤50 nM, and ≤40 nM. D ).

[0058] Experimental results show that the anti-human interleukin-23 (IL-23) monoclonal antibody of the present invention can specifically bind to the p19 subunit of human interleukin-23 (IL-23).

[0059] The anti-human interleukin-23 (IL-23) monoclonal antibody of the present invention is comparable to or superior to commercially available monoclonal antibody products in many aspects of biological activity. These biological activities include, for example, inhibiting IL-23-induced STAT3 phosphorylation in cells, inhibiting IL-17A release from mouse spleen cells induced by IL-23, and inhibiting IFN-γ release from human NK cells induced by IL-23.

[0060] In one embodiment, the amino acid sequence of the heavy chain of the anti-human interleukin-23 (IL-23) monoclonal antibody of the present invention is shown in SEQ ID NO: 11; the amino acid sequence of the light chain is shown in SEQ ID NO: 12.

[0061] SEQ ID NO: 11

[0062]

[0063] SEQ ID NO: 12

[0064]

[0065] Among them, SEQ ID NO: 11 and 12 are both humanized sequences.

[0066] In this specification, "isolated" nucleic acid means a nucleic acid molecule that has been isolated from components of its native environment. Isolated nucleic acid includes nucleic acid molecules that are normally found in cells containing nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations other than their native chromosomal locations.

[0067] In this specification, "isolated nucleic acid encoding anti-human interleukin-23 monoclonal antibody" means one or more nucleic acid molecules encoding the heavy and light chains of the antibody, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in the host cell.

[0068] In this specification, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0069] In this specification, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny (regardless of passage number). Progeny may not be identical to parental cells in terms of nucleic acid contents, but may contain mutations. Mutant progeny with the same function or biological activity selected from the initially transformed cells are included in this specification.

[0070] In this specification, "pharmaceutical composition" means an article which is in a form that enables the bioactivity of the active ingredient contained therein to exert its effect, and which does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation is to be administered.

[0071] In this specification, "pharmaceuticalally acceptable carrier" means a component of the pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0072] Example

[0073] The present invention will now be described in more detail through embodiments. It should be understood that the present invention is not limited to these embodiments.

[0074] Example 1: Preparation of anti-human interleukin-23 monoclonal antibody QX004N

[0075] Interleukin-23 (IL-23) was purchased from Shanghai Nearshore Technology Co., Ltd. for immunizing New Zealand rabbits. B-cell cloning technology was used to obtain antigen-binding specific antibody clones, and then monoclonal antibodies binding to IL-23 and exhibiting IL-23 inhibitory activity were screened. First, the cell supernatant was analyzed using Binding ELISA to select clones binding to IL-23; then, Blocking ELISA was used to select clones with IL-23 inhibitory activity. The immunization and screening processes were outsourced to a commercial company.

[0076] Five clones were selected for recombinant expression and sequencing. Clone 90# was humanized. Human IgG germline sequence homology was performed using NCBI IgBlast, and IGHV3-66*01 was selected as the heavy chain CDR transplantation template. The CDR regions of the heavy chain from clone 90# (i.e., CDR-H1 (SEQ ID No:1), CDR-H2 (SEQ ID No:2), and CDR-H3 (SEQ ID No:3)) were transplanted into the backbone region of IGHV3-66*01. IGKV1-39*01 was selected as the light chain CDR transplantation template, and the CDR regions of the light chain from clone 90# (i.e., CDR-L1 (SEQ ID No:4), CDR-L2 (SEQ ID No:5), and CDR-L3 (SEQ ID No:3)) were transplanted into the backbone region of IGHV3-66*01. No:6) was transplanted into the backbone region of IGKV1-39*01; a reverse mutation was performed at a specific site in the backbone region, mutating methionine (Met, M) at position 103 of the heavy chain CDR-H3 into leucine (Leu, L), to obtain the variable region of the monoclonal antibody QX004N of this invention. Finally, the humanized heavy chain variable region sequence is shown in SEQ ID NO: 7; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 8.

[0077] The gene of the heavy chain variable region (SEQ ID NO: 7) was obtained by PCR amplification; the gene of the light chain variable region (SEQ ID NO: 8) was obtained by PCR amplification. The heavy chain expression plasmid pHZDCH was digested with HindIII and NheI; the light chain expression plasmid pHZDCK was digested with HindIII and BsiWI; the PCR-amplified genes were inserted into the corresponding expression plasmids using Infusion recombinase to construct the heavy chain expression plasmid pHZDCH-90VH-Hu18 and the light chain expression plasmid pHZDCK-90VK-Hu9.

[0078] Figure 1 shows the results of double enzyme digestion of the plasmids detected by nucleic acid electrophoresis. Figure 1 shows the PCR amplification results of the antibody heavy chain variable region and the light chain variable region, as well as the results of double enzyme digestion of the heavy and light chain expression plasmids. The heavy and light chain plasmids are approximately 10,000 bp in size, with the light chain variable region being approximately 438 bp and the heavy chain variable region approximately 459 bp.

[0079] The correct heavy chain expression plasmid and light chain expression plasmid were co-transfected into ExpiCHO-S cells. One day before transfection, ExpiCHO-S cells were diluted to 3 × 10⁻⁶ cells / mL. 6 Cells were passaged at a density of 10 cells / ml before transfection. On the day of transfection, the cell density was diluted to 6 × 10⁻⁶ cells / ml. 6 Cells / ml, 25ml of cells were placed in a 125ml shake flask and awaited transfection. The transfection and expression process is shown in Figure 2.

[0080] Days 4-8 post-transfection, the culture supernatant was harvested and purified in one step using Protein A. The purified antibody was detected by SDS-PAGE electrophoresis and named QX004N (HZD90-32). The results of protein electrophoresis of this antibody are shown in Figure 3. Protein electrophoresis was performed using a denaturing reducing gel. The results in Figure 3 show two bands, with sizes of approximately 50 kDa and 25 kDa, which are consistent with the theoretical molecular weights of the heavy chain (49.1 kDa) and light chain (23.1 kDa).

[0081] Example 2 Equilibrium dissociation constant (K) D Determination of )

[0082] The affinity of QX004N (HZD90-32) for IL-23 was detected using a Biacore T200 instrument, with all procedures performed at 25°C. A commercial Protein A chip was used, and an appropriate amount of antibody was immobilized via a capture method to achieve an Rmax of approximately 50 RU at a capture flow rate of 10 μl / min. The antigen was serially diluted, and the flow rate was switched to 30 μl / min, flowing sequentially from low to high concentration through the reference channel and the channel containing the immobilized antibody. A buffer solution was used as a negative control. After each binding and dissociation cycle, the chip was regenerated using glycine at pH 1.5. The instrument's built-in analysis software was used to fit the 1:1 binding model in the Kinetics option, and the antibody binding rate constant k was calculated. a dissociation rate constant k d and the dissociation equilibrium constant K D value.

[0083] In addition, the affinity of QX004N (HZD90-32) was compared with that of commercially available monoclonal antibodies against IL-23, namely Guselkumab and Risankizumab. The detection methods for the known antibodies were the same as those for QX004N, and the results are shown in Table 1. Guselkumab and Risankizumab were obtained by purchasing commercially available pharmaceutical products.

[0084] Table 1. Affinity of antibody to human IL-23

[0085] Sample name k a (10 5 M -1 S -1 )k d (10 -5 S -1 )K D (10 -10M) Guselkumab5.013.190.63Risankizumab7.063.700.52QX004N(HZD90-32)3.663.501.01

[0086] The data in the table is the average value of the data obtained from testing each sample twice.

[0087] Example 3: QX004N, Guselkumab, and Risankizumab inhibit IL-23-induced HEK Blue. TM STAT3 phosphorylation activity in IL-23 cells

[0088] Leveraging HEK Blue TM Assay of IL-23 reporter gene cell lines: QX004N, Guselkumab, and Risankizumab inhibited the phosphorylation activity of the intracellular signaling molecule STAT3 mediated by IL-23. Cells in culture medium were cultured at a density of 4 × 10⁶ cells per well. 4 Cells were added to 96-well plates and cultured overnight in a CO2 incubator (37°C, 5% CO2). IL-23 was diluted to 1 ng / ml, and the antibody was diluted to serial dilutions ranging from 0 to 2 μg / ml. IL-23 and serially diluted antibody were mixed in equal volumes and incubated in a CO2 incubator (37°C, 5% CO2) for 1.5 hours. The mixture was then added to the cells and cultured in a CO2 incubator (37°C, 5% CO2) for 24 hours. The cell culture supernatant was collected and 10% QUANTI-Blue was added. TM The test reagent was reacted at 37°C for 1 hour, and then the OD630nm value was measured and a dose-response curve was plotted to analyze the neutralizing activity of the antibody. The dose-response curve is shown in Figure 4.

[0089] The results shown in Figure 4 indicate that QX004N can inhibit IL-23-induced HEK Blue. TM STAT3 phosphorylation activity in IL-23 cells, and its IC50 50 The concentration was 3.21 ng / ml; Guselkumab and Risankizumab also inhibited IL-23-induced HEK Blue. TM STAT3 phosphorylation activity in IL-23 cells, and its IC50 50 The concentrations were 6.18 ng / ml and 3.51 ng / ml, respectively, indicating that QX004N's ​​inhibitory activity against IL-23-induced signal transduction is comparable to that of Risankizumab, a commercially available monoclonal antibody against IL-23, and superior to Guselkumab.

[0090] Example 4: QX004N, Guselkumab, and Risankizumab inhibit IL-23-induced release of IL-17A from mouse spleen cells.

[0091] The inhibitory activity of QX004N, gusselukab, and risankizumab on IL-23-induced IL-17A release was determined using mouse spleen cells: Primary mouse spleen cells were obtained from mouse spleens, and the cells in the culture medium were cultured at 5 × 10⁶ cells per well. 5 Cells were added to 96-well plates. IL-23 was diluted to 10 ng / ml, and the antibody was diluted to serial dilutions ranging from 0 to 20 μg / ml. IL-23 and serially diluted antibody were mixed in equal volumes and incubated in a CO2 incubator (37°C, 5% CO2) for 1.5 hours. The mixture was then added to the cells and incubated in a CO2 incubator (37°C, 5% CO2) for 48 hours. The cell culture supernatant was collected, and the expression level of IL-17A was detected using an ELISA kit to plot a dose-response curve, which was then used to analyze the neutralizing activity of the antibody. The dose-response curve is shown in Figure 5.

[0092] The results shown in Figure 5 indicate that QX004N can inhibit IL-23-induced IL-17A release from mouse spleen cells, with an IC50 value of [missing information]. 50 The concentration was 11.7 ng / ml; Guselkumab and Risankizumab also inhibited IL-23-induced release of IL-17A from mouse spleen cells, with an IC50 of 11.7 ng / ml. 50 The concentrations were 13.5 ng / ml and 8.43 ng / ml, respectively, indicating that QX004N has a strong inhibitory activity against IL-23-induced release of IL-17A from mouse spleen cells, which is comparable to that of existing commercial products (Guselkumab and Risankizumab).

[0093] Example 5: QX004N, Guselkumab, and Risankizumab inhibit IL-23-induced IFN-γ release from human NK cells.

[0094] The inhibitory activity of QX004N, gusselukab, and risankizumab on IL-23-induced IFN-γ release was determined using human NK cells. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers using density gradient centrifugation, and natural killer cells (NK cells) were then sorted from PBMCs using magnetic bead sorting. Cells were activated and cultured for 3 days in a CO2 incubator (37℃, 5% CO2) with 100 U / ml IL-2. Then, 20 ng / ml IL-18 was added to the culture medium, and the cells were cultured at a density of 1 × 10⁶ cells / well. 5Cells were added to 96-well plates. IL-23 was diluted to 10 ng / ml, and the antibody was diluted to serial dilutions ranging from 0 to 5 μg / ml. IL-23 and serially diluted antibody were mixed in equal volumes and incubated in a CO2 incubator (37°C, 5% CO2) for 1.5 hours. The mixture was then added to the cells and incubated in a CO2 incubator (37°C, 5% CO2) for 48 hours. The cell culture supernatant was collected, and the expression level of IFN-γ was detected using an ELISA kit to plot a dose-response curve. The neutralizing activity of the antibody was then analyzed. The dose-response curve is shown in Figure 6.

[0095] The results shown in Figure 6 indicate that QX004N can inhibit IL-23-induced IFN-γ release activity from human NK cells, and its IC50 value is [missing information]. 50 The concentration was 10.4 ng / ml; Guselkumab and Risankizumab also inhibited IL-23-induced IFN-γ release from human NK cells, with an IC50 of 10.4 ng / ml. 50 The concentrations were 16.8 ng / ml and 11.1 ng / ml, respectively, indicating that QX004N has a stronger inhibitory activity against IL-23-induced IFN-γ release from human NK cells than the currently commercialized product Guselkumab, and its activity is comparable to that of Risankizumab.

[0096] As can be seen from Examples 3-5 above, in terms of the three measured cellular-level biological activities, QX004N is superior to Guselkumab, and comparable to Risankizumab. Given that Risankizumab... It has been proven in clinical trials to have significant therapeutic effects on moderate to severe plaque psoriasis, and QX004N is also expected to show good clinical effects in the prevention and treatment of related diseases.

[0097] Example 6: Specific detection of QX004N, Guselkumab, and Risankizumab binding to the IL-23 p19 subunit

[0098] The binding of QX004N, Guselkumab, and Risankizumab to the p40 subunit of IL-23 was determined using a Binding ELISA method. IL-23-p40 was diluted to 2 μg / ml and added to an ELISA plate, which was then incubated overnight at 4°C. After blocking, a series of antibody dilutions (0–5 μg / ml) and Goat-anti-human IgG secondary antibody were added and incubated. Finally, substrate was added for color development, and the reaction was terminated with stop solution. After termination, the absorbance at 450 nm was read using a microplate reader (650 nm absorbance was used as a reference), and a dose-response curve was plotted to analyze the antibody binding specificity. The dose-response curve is shown in Figure 7.

[0099] The results shown in Figure 7 indicate that the IL-23 p40-specific antibody QX001S can bind to the p40 subunit, while QX004N, Guselkumab, and Risankizumab do not bind to the p40 subunit of IL-23, suggesting that QX004N, Guselkumab, and Risankizumab specifically bind to the p19 subunit of IL-23.

[0100] Example 7: Investigating the difference in binding of QX004N and Risankizumab to IL-23p19 using point mutations.

[0101] Mutation primers were designed using PCR to construct p19 plasmids containing five single-point mutations, as shown in Table 2. The p19 mutant plasmid was co-transfected with the p40 plasmid into 293F cells to prepare cell supernatants containing the IL-23 mutant. The protein concentration of the IL-23 mutant in the cell supernatant was first quantified using quantitative ELISA. Then, conjugation ELISA was used to detect the EC50 of QX004N and Risankizumab with different IL-23 mutants. 50 value.

[0102] The concentration of cell supernatant containing the IL-23 mutant was quantified using a Binding ELISA method: Anti-p40 antibody was diluted to 2 μg / ml and added to an ELISA plate, which was then incubated overnight at 4°C. After blocking, purified IL-23 diluted to known concentrations ranging from 0 to 50 ng / ml, along with serially diluted IL-23 mutant cell supernatant, was added and incubated. Biotin-labeled anti-His antibody and SA-HRP were then added and incubated separately. Finally, substrate was added for color development, and the reaction was terminated with stop solution. After termination, the absorbance at 450 nm was read using an ELISA reader (650 nm absorbance was used as a reference), and a dose-response curve of the known concentration of purified IL-23 was plotted as a standard curve. The concentration of the IL-23 mutant cell supernatant was then calculated based on the standard curve and the OD value of the IL-23 mutant cell supernatant.

[0103] The binding of QX004N and Risankizumab to the IL-23 mutant was detected using a Binding ELISA method: QX004N and Risankizumab were diluted to 1 μg / ml and added to ELISA plates, and incubated overnight at 4°C. After blocking, serially diluted IL-23 mutant cell supernatant at concentrations ranging from 0 to 0.5 μg / ml were added and incubated. Then, biotin-labeled anti-His antibody and SA-HRP were added for incubation, respectively. Finally, substrate was added for color development, and the reaction was terminated with stop solution. After termination, the absorbance at 450 nm was read in an ELISA reader (650 nm absorbance was used as a reference), a dose-response curve was plotted, and EC50 was calculated. 50 Value. If the EC of the IL-23 mutant is... 50 If the value is more than 10 times larger than that of natural IL-23, it indicates that the mutation site corresponds to an antibody-binding epitope, EC. 50 The results are shown in Table 2.

[0104] The results shown in Table 2 indicate that QX004N and Risankizumab have different epitopes binding to IL-23p19, and W137 is a key epitope for QX004N. Jutta Yehudi Bloch et al. confirmed that W137 is one of the key sites for IL-23p19 to bind to the IL-23 receptor (IL-23R).

[0105] Table 2 shows the antibody binding to different IL-23 mutants detected by ELISA.

[0106]

[0107] ND indicates weak binding, EC 50The value is inaccurate.

[0108] In summary, the monoclonal antibody described in this invention can inhibit STAT3 phosphorylation activity, inhibit IL-23-induced release of IL-17A from mouse spleen cells, and inhibit IL-23-induced release of IFN-γ from human NK cells. Its inhibitory activity is superior to that of currently commercialized products, namely Guselkumab, and comparable to that of Risankizumab. Regarding the IL-23-induced release of IL-17A from mouse spleen cells, the monoclonal antibody described in this invention has an inhibitory activity comparable to that of Guselkumab and Risankizumab.

[0109] The monoclonal antibody described in this invention, like currently commercialized products (Guselkumab and Risankizumab), can specifically bind to the p19 subunit of IL-23. However, the monoclonal antibody described in this invention has a different epitope binding to IL-23 p19 compared to Guselkumab and Risankizumab. W137 is a key epitope of the monoclonal antibody described in this invention, but it is not a key epitope of Guselkumab or Risankizumab (for epitope studies of Guselkumab, see International Patent Application Publication WO2007076524A2; for epitope studies of Risankizumab, see Sanjaya et al., Selective targeting of the IL23 pathway Generation and characterization of a novel high affinity humanized anti-IL23A antibody, mAbs, Volume 7, Issue 4, 2015).

[0110] Furthermore, due to Risankizumab It has been approved for marketing in Japan, the United States, and the European Union, and clinical trial results show that it is more effective than Johnson & Johnson's blockbuster anti-inflammatory drug in treating moderate to severe plaque psoriasis. (ustekinumab) and AbbVie's best-selling anti-inflammatory drug (adalimumab), therefore, the monoclonal antibody described in this invention is expected to show good clinical efficacy in the prevention and treatment of related diseases.

Claims

1. An isolated anti-human interleukin-23 monoclonal antibody, which comprises three heavy-chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light-chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein: (a) The amino acid sequence of CDR-H1 is as shown in SEQ ID NO: 1; (b) The amino acid sequence of CDR-H2 is as shown in SEQ ID NO: 2; (c) The amino acid sequence of CDR-H3 is as shown in SEQ ID NO: 3; (d) The amino acid sequence of CDR-L1 is as shown in SEQ ID NO: 4; (e) The amino acid sequence of CDR-L2 is as shown in SEQ ID NO: 5; and (f) The amino acid sequence of CDR-L3 is as shown in SEQ ID NO:

6.

2. The monoclonal antibody according to claim 1, which comprises a heavy-chain variable region and a light-chain variable region, wherein, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7; and, The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

8.

3. An isolated nucleic acid encoding the monoclonal antibody according to claim 1 or 2.

4. A host cell comprising the nucleic acid according to claim 3.

5. A method for producing a monoclonal antibody, the method comprising culturing the host cell according to claim 4 to produce the monoclonal antibody according to claim 1 or 2.

6. A pharmaceutical composition comprising the monoclonal antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, which is used for treating diseases related to IL-23-mediated signal transduction.

8. The pharmaceutical composition according to claim 7, wherein, The diseases related to IL-23-mediated signal transduction are rheumatoid arthritis, juvenile rheumatoid arthritis, systemic-onset juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, gastric ulcer, inflammatory bowel disease, ulcerative colitis, acute pancreatitis, primary biliary cirrhosis, Hashimoto's thyroiditis, systemic lupus erythematosus, iridocyclitis, uveitis, optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis / Wegener's granulomatosis, allergic / atopic diseases, asthma, allergic rhinitis, eczema, adult respiratory distress syndrome, allergic contact dermatitis, vitiligo, psoriasis, alopecia areata, pemphigus, scleroderma, allergic conjunctivitis, allergic pneumonia, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, Graves' disease, Raynaud's disease, type B insulin-resistant diabetes, myasthenia gravis, nephrotic syndrome, nephritis, glomerulonephritis, and / or acute renal failure.

9. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a drug for treating diseases related to IL-23-mediated signal transduction.

10. The use according to claim 9, wherein, The diseases related to IL-23-mediated signal transduction are selected from rheumatoid arthritis, juvenile rheumatoid arthritis, systemic-onset juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, gastric ulcer, inflammatory bowel disease, ulcerative colitis, acute pancreatitis, primary biliary sclerosis, Hashimoto's thyroiditis, systemic lupus erythematosus, iridocyclitis, uveitis, optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis / Wegener's granulomatosis, allergic / atopic diseases, asthma, allergic rhinitis, eczema, adult respiratory distress syndrome, allergic contact dermatitis, vitiligo, psoriasis, alopecia areata, pemphigus, scleroderma, allergic conjunctivitis, allergic pneumonia, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, Graves' disease, Raynaud's disease, type B insulin-resistant diabetes, myasthenia gravis, nephrotic syndrome, nephritis, glomerulonephritis, and / or acute renal failure.