Patent Description:
IL-<NUM> is mainly produced by activated dendritic cells, macrophages and monocytes, etc. It is a member of the IL-<NUM> heterodimer cytokine family, mainly composed of IL-23p19 and IL-<NUM>/IL-23p40 subunits. IL-<NUM> receptors comprise <NUM> subunits which are IL-<NUM> receptor β1 and IL-<NUM> receptor. IL-<NUM> activates downstream signal pathways to exert biological functions by expressing receptors IL-23R and IL-12R β1 on the surface of T cells, NK cells, and monocyte macrophages/dendritic cells. IL-<NUM> mainly acts on Th17 cells and induces them to produce proinflammatory cytokines such as IL-17A, IL-17F, IL-<NUM>, IL-<NUM>, etc., and plays an important role in autoimmune and inflammatory diseases such as psoriasis, psoriatic arthritis, multiple sclerosis, Crohn's disease, inflammatory bowel disease, etc..

IL-<NUM> mediated signal transduction and biological effects are related to many types 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 biliary cirrhosis, Hashimoto's thyroiditis, systemic lupus erythematosus, iridocyclitis, uveitis, optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegener's granuloma, allergic/atopic disease, 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 resistance diabetes, myasthenia gravis, nephrotic syndrome, nephritis, glomerulonephritis and/or acute renal failure, etc..

Guselkumab (trade name Tremfya) and Risankizumab (trade name SKYRIZI), monoclonal antibody drugs targeting IL-<NUM> developed by Johnson & Johnson and Abbvie respectively, have been approved by the US FDA to treat psoriasis and psoriatic arthritis, and to carry out phase III clinical research on Crohn's disease and inflammatory bowel disease. <CIT> relates to humanized IL-<NUM> monoclonal antibodies, namely MAbQF20 and MAbQF37, binding to the p19 subunit of IL-<NUM>.

The object of the present application is to provide a new anti-human interleukin-<NUM> (hIL-<NUM>) monoclonal antibody, a pharmaceutical composition comprising the monoclonal antibody and pharmaceutical use of the monoclonal antibody.

That is, the present application relates to the following aspects.

The present invention relates to an isolated anti-human interleukin <NUM> monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein,.

The interleukin23 monoclonal antibody thus 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:.

The present invention also relates to an isolated nucleic acid, encoding the monoclonal antibody of the present invention.

The present invention relates also to a host cell, comprising the nucleic acid as defined above.

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 one of a variety of host cells can be used. In one embodiment, the host cell is a prokaryotic cell, for example, Escherichia coli (E. In another embodiment, the host cell is a eukaryotic cell, for example, a mammalian cell such as a Chinese hamster ovary (CHO) cell.

In another aspect, the present invention relates to a method for producing a monoclonal antibody, comprising culturing the host cell as disclosed above to produce the monoclonal antibody of the present invention.

The method includes expressing a recombinant vector encoding the anti-human interleukin <NUM> monoclonal antibody in a suitable host cell, thereby producing the monoclonal antibody. In certain embodiments, the method includes culturing a host cell comprising a nucleic acid encoding the anti-human interleukin <NUM> monoclonal antibody, thereby expressing the nucleic acid. The method may further include recovering the anti-human interleukin <NUM> monoclonal antibody from a host cell culture or host cell culture medium.

The present invention further relates to a pharmaceutical composition, comprising the monoclonal antibody of the present invention and a pharmaceutically acceptable carrier.

The pharmaceutical composition may further comprise an additional therapeutic agent (for example, a different anti-human interleukin23 (hIL-<NUM>) antibody).

The present invention further relates to the pharmaceutical composition as disclosed herein for use for treatment of a disease related to IL-<NUM> mediated signal transduction.

The disease related to IL-<NUM> mediated signal transduction is selected from the group consisting of: 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, systemic vasculitis/Wegener's granuloma, allergic/atopic disease, 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 resistance diabetes, myasthenia gravis, nephrotic syndrome, nephritis, glomerulonephritis and/or acute renal failure.

Preferably, the systemic vasculitis is Wegener's granuloma.

The present application provides a new anti-human interleukin <NUM> (hIL-<NUM>) monoclonal antibody. Compared with the existing anti-human interleukin <NUM> monoclonal antibodies (Guselkumab and Risankizumab), it has a comparable affinity for binding to hIL-<NUM>, but its antagonistic activity at the cellular level is superior to that of Guselkumab, and is comparable to that of Risankizumab.

It should be noted that Risankizumab (SKYRIZI®) has been approved for marketing in Japan, the United States and the European Union, and the clinical trial results show that its therapeutic effect on moderate to severe plaque psoriasis is superior to that of Johnson & Johnson's blockbuster anti-inflammatory drug STELARA® (ustekinumab) and AbbVie's best-selling anti-inflammatory drug HUMIRA® (adalimumab).

The monoclonal antibody of the present application shows antagonistic activity comparable to Risankizumab at the cell level, and it is expected to show good clinical effects in the prevention and treatment of related diseases.

The scientific and technological terms mentioned in this specification have the same meanings as those commonly understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

In general, the terms used in this specification have the following meanings.

In this specification, "isolated" antibody is an antibody that has been separated from a component of its natural environment. In certain embodiments, the antibody is purified to a purity greater than <NUM>% or <NUM>%, which is determined by, for example, electrophoresis (for example, SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (for example, ion exchange or reverse phase HPLC). For a review of methods for evaluating antibody purity, see, for example, <NPL>).

In this specification, "monoclonal antibody" means an antibody derived from a population of substantially homologous antibodies, that is, each antibody constituting the population is the same and/or binds to the same epitope, except for possible variant antibodies (for example, comprising naturally occurring mutations or produced in the production process of monoclonal antibody products), such variants are usually present in trace amounts. Unlike polyclonal antibody products that generally comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody of the monoclonal antibody product is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the characteristic that the antibody is derived from a substantially homologous antibody population, and should not be interpreted as requiring the production of the antibody by any specific method. For example, the monoclonal antibody to be used according to the present application can be prepared by a variety of technologies; the technologies include, but are not limited to, a hybridoma method, a recombinant DNA method, a phage display method, and a method using a transgenic animal comprising all or part of the human immunoglobulin locus. Such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

In this specification, "affinity" means the strength of the sum of non-covalent interactions between a single binding site of a molecule (for example, an antibody) and its binding partner (for example, an antigen). Unless otherwise noted, the "binding affinity" used in this specification means the inherent binding affinity reflecting the <NUM>:<NUM> interaction between members of a binding pair (for example, antibody and antigen). The affinity of molecule X to its partner Y can usually be denoted by the equilibrium dissociation constant (KD). Affinity can be measured by common methods known in the art.

In this specification, human interleukin <NUM> (hIL-<NUM>) represents a human derived protein, which is a heterodimer consisting of p19 and p40 subunits. The amino acid sequence of p19 is represented by SEQ ID NO: <NUM>, and the amino acid sequence of p40 is represented by SEQ ID NO: <NUM>, wherein the underlined part represents a signal peptide.

In this specification, "anti-human interleukin <NUM> monoclonal antibody" means a monoclonal antibody that can bind to human interleukin <NUM> with sufficient affinity, so that the monoclonal antibody can be used as a diagnostic agent and/or therapeutic agent targeting human interleukin <NUM>.

The anti-human interleukin <NUM> (IL-<NUM>) monoclonal antibody in the present application does not bind to a target unrelated protein. Herein, "unrelated protein" refers to a protein except for the target human interleukin <NUM>; Herein, "non-binding" means that when the binding ability of the anti-human interleukin-<NUM> (IL-<NUM>) monoclonal antibody of the present application to its target human interleukin-<NUM> is <NUM>%, the binding ability of the anti-human interleukin-<NUM> (IL-<NUM>) monoclonal antibody of the present application to the unrelated proteins is less than <NUM>%, such as <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>.

The anti-human interleukin <NUM> (IL-<NUM>) monoclonal antibody of the present application may not bind to interleukin <NUM> of other animal species. Herein, "other animal species" refers to other animal species except for human beings, such as rhesus monkey, cynomolgus monkey, rat, mouse, etc.; Herein, "non-binding" means that when the binding ability of the anti-human interleukin-<NUM> (IL-<NUM>) monoclonal antibody of the present application to its target human interleukin-<NUM> is <NUM>%, the binding ability of the anti-human interleukin-<NUM> (IL-<NUM>) monoclonal antibody of the present application to interleukin-<NUM> of other animal species is less than <NUM>%, such as <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>.

The anti-human interleukin-<NUM> (IL-<NUM>) monoclonal antibody of the present application has an equilibrium dissociation constant (KD) of ≤<NUM>, ≤<NUM>, ≤<NUM> or ≤<NUM>.

The experimental results show that the anti-human interleukin-<NUM> (IL-<NUM>) monoclonal antibody of the present application can specifically bind to the p19 subunit of human interleukin-<NUM> (IL-<NUM>).

The anti-human interleukin <NUM> (IL-<NUM>) monoclonal antibody of the present application is comparable to or superior to the marketed similar monoclonal antibody products in many biological activities. The biological activity, such as activity of inhibiting phosphorylation of STAT3 in cells induced by IL-<NUM>, activity of inhibiting the release of IL-17A from mouse spleen cells induced by IL-<NUM>, and activity of inhibiting the release of IFN-γ from human NK cells induced by IL-<NUM>.

In one embodiment, the amino acid sequence of the heavy chain of the anti-human interleukin-<NUM> (IL-<NUM>) monoclonal antibody of the present application is represented by SEQ ID NO: <NUM>; the amino acid sequence of the light chain is represented by SEQ ID NO: <NUM>.

Particularly, both of SEQ ID NO: <NUM> and SEQ ID NO: <NUM> are humanized sequences.

In this specification, "isolated" nucleic acid means a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid comprises a nucleic acid molecule contained in a cell that usually contains a nucleic acid molecule, but the nucleic acid molecule is present outside the chromosome or in a chromosome position different from its natural chromosome position.

In this specification, "isolated nucleic acid encoding anti-human interleukin <NUM> monoclonal antibody" means one or more nucleic acid molecules encoding heavy chains and light chains of the antibody, including such nucleic acid molecules in a single vector or separated vectors, and such nucleic acid molecules present in one or more positions in host cell.

In this specification, "vector" means a nucleic acid molecule capable of amplifying another nucleic acid linked to it. The term includes a vector as a self-replicating nucleic acid structure and a vector integrated into the genome of a host cell into which it has been introduced. Certain vectors can guide the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors".

In this specification, "host cell", "host cell line" and "host cell culture" are used interchangeably, and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom (regardless of the number of passages). The progeny may not be identical with the parent cell in terms of nucleic acid content, but may comprise a mutation. The mutant progeny with the same function or biological activity screened or selected for initially transformed cells are included in this specification.

In this specification, "pharmaceutical composition" means a product that presents in a form that enables the biological activity of the active ingredient contained therein to take effects, and the composition does not contain additional components with unacceptable toxicity to the subject to which the preparation is to be administered.

In this specification, "pharmaceutically acceptable carrier" means an ingredient other than the active ingredient in the pharmaceutical composition, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

Hereinafter, the present application will be described in more detail through examples.

Human interleukin <NUM> (IL-<NUM>) was purchased from Shanghai Novo protein Technology Co. to immunize New Zealand rabbits. Antigen binding specific antibody clones were obtained through B cell cloning technology, and then monoclonal antibodies binding to IL-<NUM> and having inhibitory activity against IL-<NUM> were screened out. Firstly, the cell supernatant was detected by Binding ELISA, and clones binding to IL-<NUM> were selected; then clones having inhibitory activity against IL-<NUM> were selected by using Blocking ELISA detection. The above immunization and screening processes were entrusted to commercial companies.

Five clones were selected for recombinant expression and sequencing. The clone <NUM># was humanized. NCBI IgBlast was used to perform homology comparison of human IgG germline sequences, IGHV3-<NUM>*<NUM> was selected as the template for heavy chain CDR grafting, and the heavy chain CDR regions of the clone <NUM># (i.e., CDR-H1 (SEQ ID No: <NUM>), CDR-H2 (SEQ ID No: <NUM>) and CDR-H3 (SEQ ID No: <NUM>)) were grafted into the framework region of IGHV3-<NUM>*<NUM>; IGKV1-<NUM>*<NUM> was selected as template for the light chain CDR grafting, and the light chain CDR regions of the clone <NUM># (i.e. CDR-L1 (SEQ ID No: <NUM>), CDR-L2 (SEQ ID No: <NUM>) and CDR-L3 (SEQ ID No: <NUM>)) were grafted into the framework region of IGKV1-<NUM>*<NUM>; Reverse mutation was carried out at the specific site of the framework region, and the methionine (Met, M) at position <NUM> in heavy chain CDR-H3 was mutated to leucine (Leu, L), to obtain the variable region of the monoclonal antibody QX004N of the present application. Finally, the amino acid sequence of the humanized heavy chain variable region is represented by SEQ ID NO: <NUM>; the amino acid sequence of the humanized light chain variable region is represented by SEQ ID NO: <NUM>.

The gene of the above heavy chain variable region (SEQ ID NO: <NUM>) was obtained by PCR amplification; the gene of the light chain variable region (SEQ ID NO: <NUM>) 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 with Infusion recombinant enzyme to construct the heavy chain expression plasmid pHZDCH-90VH-Hu18 and the light chain expression plasmid pHZDCK-90VK-Hu9.

The results of the double digestion of plasmids detected by nucleic acid electrophoresis are shown in <FIG>. According to the results in <FIG>, it can be seen the PCR amplification results of the heavy chain variable region and light chain variable region of the antibody and the results of double digestion of the heavy chain and light chain expression plasmids, wherein, the size of the plasmids of the heavy chains and light chains is about <NUM> bp, the light chain variable region is about <NUM> bp, and the heavy chain variable region is about <NUM> bp.

The heavy chain expression plasmid and light chain expression plasmid with correct sequence were co-transfected into ExpiCHO-S cells. The day before transfection, ExpiCHO-S cells were diluted to <NUM> × <NUM><NUM> cells/ml for passage before transfection. On the day of transfection, the cell density was diluted to <NUM> × <NUM><NUM> cells/ml, and <NUM> cells were placed in <NUM> shake flask, waiting for transfection. The process of transfection and expression is shown in <FIG>.

The culture supernatant was harvested <NUM>-<NUM> days after transfection, and was further purified with ProteinA in one step. The purified antibody was detected by SDS-PAGE electrophoresis, and named as QX004N (HZD90-<NUM>). The result of the detection of detecting the antibody by protein electrophoresis is shown in <FIG>. The protein electrophoresis was detected using denatured reducing gel. The result in <FIG> shows that there are two bands, and the sizes of the two bands are about <NUM> kDa and <NUM> kDa respectively, which are consistent with the theoretical molecular weights of the heavy chain (<NUM> kDa) and light chain (<NUM> kDa).

The affinity of QX004N (HZD90-<NUM>) binding to IL-<NUM> was detected using BiacoreT200, and all processes were carried out at <NUM>. Commercialized Protein A chip was used to immobilize an appropriate amount of antibody by capture method, the Rmax was about <NUM> RU and the capture flow rate was 10µl/min. The antigen was diluted by gradient, and the flow rate of the instrument was switched to 30µl/min. The diluted antigen flowed through the reference channel and the channel of immobilized antibody in sequence from low to high concentration, and flowed through the buffer as negative control. The chip was regenerated with glycine at pH <NUM> after each binding and dissociation. Fitting was performed with the <NUM>:<NUM> binding model in the Kinetics option using the build-in software in the instrument, and the association rate constant ka, dissociation rate constant kd and equilibrium dissociation constant KD of the antibody were calculated.

In addition, the affinity of QX004N (HZD90-<NUM>) was compared with the currently commercialized monoclonal antibodies against IL-<NUM>, i.e., Guselkumab and Risankizumab. The detection method for known antibodies was the same as the detection method for QX004N. The results are shown in Table <NUM>, wherein, Guselkumab and Risankizumab were obtained by purchasing commercially available drugs.

The data in the table were: obtained by calculating the average value of the test results after each sample was tested twice.

The activities of QX004N, Guselkumab and Risankizumab inhibiting phosphorylation of intracellular signal molecule STAT3 induced by IL-<NUM> were detected using HEK Blue™ IL-<NUM> cell reporter gene cell line: cells in culture media were added into <NUM>-well plate at <NUM>×<NUM><NUM> cells per well, and cultured overnight in CO<NUM> incubator (<NUM>, <NUM>% CO<NUM>). IL-<NUM> was diluted to 1ng/ml, the antibody was diluted to a series of dilutions with a concentration range of <NUM> to <NUM>µg/ml, and IL-<NUM> was mixed with the gradient diluted antibody in equal volumes, incubated in CO<NUM> incubator (<NUM>, <NUM>% CO<NUM>) for <NUM> hours, then the mixed solutions were added to the cells and cultured in CO<NUM> incubator (<NUM>, <NUM>% CO<NUM>) for <NUM> hours. The cell culture supernatant was collected and added with <NUM>% QUANTI-Blue™ detection reagent to react at <NUM> for <NUM> hour, then the OD value at <NUM> was detected and the dose effect curve was plotted to analyze the neutralization activity of the antibody. The dose effect curve is shown in <FIG>.

<FIG> shows that QX004N can inhibit phosphorylation activity of STAT3 in HEK Blue™ IL-<NUM> cells induced by IL-<NUM>, with an IC<NUM> value of <NUM>. 21ng/ml; Guselkumab and Risankizumab can also inhibit phosphorylation activity of STAT3 in HEK Blue™ IL-<NUM> cells induced by IL-<NUM>, with IC<NUM> values of <NUM>. 18ng/ml and <NUM>. 51ng/ml, respectively, indicating that the activity of QX004N inhibiting signal transduction induced by IL-<NUM> is comparable to that of the commercialized monoclonal antibody against IL-<NUM> (i.e., Risankizumab), and is superior to that of Guselkumab.

The activities of QX004N, Guselkumab and Risankizumab inhibiting release of IL-17A induced by IL-<NUM> were tested using mouse spleen cells: primary mouse spleen cells were obtained from mouse spleen, and cells in the culture media were added into <NUM>-well plate at <NUM> × <NUM><NUM> cells per well. IL-<NUM> was diluted to 10ng/ml, the antibody was diluted to a series of dilutions with a concentration range of <NUM> to <NUM>µg/ml, and IL-<NUM> was mixed with the gradient diluted antibody in equal volumes, incubated in CO<NUM> incubator (<NUM>, <NUM>% CO<NUM>) for <NUM> hours, then the mixed solution was added to the cells and cultured in CO<NUM> incubator (<NUM>, <NUM>% CO<NUM>) for <NUM> hours. The cell culture supernatant was collected to detect the expression amount of IL-17A with ELISA Kit and the dose effect curve was plotted to analyze the neutralization activity of the antibody. The dose effect curve is shown in <FIG>.

<FIG> shows that QX004N can inhibit the activity of the release of IL-17A from mouse spleen cells induced by IL-<NUM>, with an IC<NUM> value of <NUM>. 7ng/ml; Guselkumab and Risankizumab can also inhibit the activity of the release of IL-17A from mouse spleen cells induced by IL-<NUM>, with IC<NUM> values of <NUM>. 5ng/ml and <NUM>. 43ng/ml, respectively, indicating that the activity of QX004N inhibiting release of IL-17A from mouse spleen cells induced by IL-<NUM> is strong, which is comparable to the existing commercialized products (Guselkumab and Risankizumab).

The activities of QX004N, Guselkumab and Risankizumab inhibiting release of IFN-γ induced by IL-<NUM> were detected using human NK cells: peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers by density gradient centrifugation, and natural killer cells (NK cells) were separated from PBMCs by magnetic bead sorting. 100U/ml of IL-<NUM> was added into CO<NUM> incubator (<NUM>, <NUM>% CO<NUM>) for activation and culture for <NUM> days. 20ng/ml of IL-<NUM> was added into the culture media, then the cells in the culture media were added into <NUM>-well plate at <NUM>×<NUM><NUM>cells per well. IL-<NUM> was diluted to 10ng/ml, the antibody was diluted to a series of dilutions with a concentration range of <NUM> to 5µg/ml, and IL-<NUM> was mixed with the gradient diluted antibody in equal volume, incubated in CO<NUM> incubator (<NUM>, <NUM>% CO<NUM>) for <NUM> hours, then the mixed solution was added to the cells and cultured in CO<NUM> incubator (<NUM>, <NUM>% CO<NUM>) for <NUM> hours. The cell culture supernatant was collected to detect the expression amount of IFN-γ with ELISA Kit and the dose effect curve was plotted to analyze the neutralizing activity of the antibody. The dose effect curve is shown in <FIG>.

<FIG> shows that QX004N can inhibit the activity of release of IFN-γ from human NK cells induced by IL-<NUM>, with an IC<NUM> value of <NUM>. 4ng/ml; Guselkumab and Risankizumab can also inhibit the activity of the release of IFN-γ from human NK cells induced by IL-<NUM>, with IC<NUM> values of <NUM>. 8ng/ml and <NUM>. 1ng/ml, respectively, indicating that the activity of QX004N inhibiting the release of IFN-γ from human NK cells induced by IL-<NUM> is stronger than that of the currently commercialized product, Guselkumab, and is comparable to that of Risankizumab.

It can be seen from above Examples <NUM> to <NUM> that, QX004N is superior to Guselkumab and comparable to Risankizumab in three measured biological activities at the cellular level. Whereas it has been proved in clinical trials that Risankizumab (SKYRIZI®) has significant therapeutic effect on moderate to severe plaque psoriasis, QX004N is also expected to show good clinical effect in the prevention and treatment of related diseases.

The binding of QX004N, Guselkumab and Risankizumab to the p40 subunit of IL-<NUM> was detected by Binding ELISA. IL-<NUM>-p40 was diluted to 2µg/ml, added to the ELISA plate, and coated overnight at <NUM>. After blocking, a series of antibody dilutions diluted to a concentration ranging from <NUM> to 5µg/ml and Goat-anti-human IgG secondary antibody were added respectively for incubation, finally substrate was added for color development, and the termination solution was added to terminate the color reaction. After termination, the ELISA plate was placed in a microplate reader to read the absorption value at <NUM> (the absorption value at <NUM> was taken as reference), and the dose effect curve was plotted to analyze the binding specificity of the antibody. The dose effect curves are shown in <FIG>.

<FIG> shows that the IL-<NUM> p40 specific antibody QX001S can bind to the p40 subunit, and QX004N, Guselkumab and Risankizumab do not bind to the p40 subunit of IL-<NUM>, indicating that QX004N, Guselkumab and Risankizumab specifically bind to the p19 subunit of IL-<NUM>.

Mutation primers were designed through PCR method. Five p19 plasmids comprising single point mutation were constructed respectively, as shown in Table <NUM>. The p19 mutant plasmid was co-transfected with p40 plasmid into 293F cells to prepare a cell supernatant containing the IL-<NUM> mutant. The protein concentration of IL-<NUM> mutants in the cell supernatants was quantified by quantitative ELISA, and then the EC<NUM> values of QX004N, Risankizumab and different IL-<NUM> mutants were detected by Binding ELISA.

Concentration of the cell supernatant comprising IL-<NUM> mutant was quantified by Binding ELISA: anti-p40 antibody was diluted to 2µg/ml and added to the ELISA plate, and coated overnight at <NUM>. After blocking, purified IL-<NUM> diluted to a known concentration ranging from <NUM> to 50ng/ml and the gradient diluted cell supernatant comprising IL-<NUM> mutant was added for incubation. Then Biotin labeled anti-His antibody and SA-HRP were added respectively to incubation, finally the substrate was added for color development, and the termination solution was added to terminate the color reaction. After termination, the ELISA plate was placed in a microplate reader to read the absorption value at <NUM> (the absorption value at <NUM> was taken as reference), and the dose effect curve of purified IL-<NUM> with known concentration was plotted as the standard curve, and then the concentration of cell supernatant of IL-<NUM> mutant was calculated according to the standard curve and the OD value of cell supernatant of IL-<NUM> mutant.

The binding of QX004N and Risankizumab to IL-<NUM> mutant was detected by Binding ELISA: QX004N and Risankizumab were respectively diluted to <NUM>µg/ml and added to the ELISA plate, and coated overnight at <NUM>. After blocking, a series of cell supernatant dilutions of IL-<NUM> mutant diluted to a concentration ranging from <NUM> to <NUM>. 5µg/ml for incubation. Then, Biotin labeled anti-His antibody and SA-HRP were added respectively for incubation, finally the substrate was added for color development, and the termination solution was added to terminate the color reaction. After termination, the ELISA plate was placed in a microplate reader to read the absorption value at <NUM> (the absorption values at <NUM> was taken as reference), and the dose effect curve was plotted and the EC<NUM> value was calculated. If the EC<NUM>value of the IL-<NUM> mutant is more than <NUM> times higher than that of the natural IL-<NUM>, it indicates that the site corresponding to the mutation is an antibody binding epitope. The EC<NUM> results are shown in Table <NUM>.

The result in Table <NUM> shows that the epitopes for QX004N and Risankizumab binding to IL-<NUM> p19 are different, and W137 is the key epitope for QX004N. Jutta Schröder and Yehudi Bloch et al. respectively confirmed that W137 is one of the key sites for IL-23p19 binding to IL-<NUM> receptor (IL-23R).

In conclusion, the monoclonal antibody of the present application can inhibit the STAT3 phosphorylation activity, IL-17A release activity of mouse spleen cells induced by IL-<NUM>, and IFN-γ release activity of human NK cells induced by IL-<NUM>. Its inhibitory activity is superior to that of the currently commercialized product (i.e., Guselkumab), and is comparable to that of Risankizumab. For IL-17A release activity of mouse spleen cells induced by IL-<NUM>, the activity of the monoclonal antibody of the present application inhibiting the release of IL-17A from the mouse spleen cells induced by IL-<NUM> is comparable to that of Guselkumab and Risankizumab.

Like the currently commercialized products (Guselkumab and Risankizumab), the monoclonal antibody of the present application can specifically bind to the p19 subunit of IL-<NUM>. However, the monoclonal antibody of the present application is different from Guselkumab and Risankizumab in binding epitopes of IL-23p19. W137 is the key epitope for the monoclonal antibody of the present application, but not the key epitope for Guselkumab or Risankizumab (the epitope research of Guselkumab can be found in the international patent publication <CIT>; the epitope research of Risankizumab can be found in "<NPL>").

Claim 1:
An isolated anti-human interleukin <NUM> monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, wherein
the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO: <NUM>; and,
the amino acid sequence of the light chain variable region is represented by SEQ ID NO: <NUM>.