CN114395041A - Method for preparing anti-IL-12 and/or IL-23 monoclonal antibody - Google Patents

Abstract

The invention relates to a method for preparing anti-IL-12 and/or IL-23 monoclonal antibody, in particular to a method for preparing Usunitumumab. Compared with antibodies expressed by SP2/0 cells and CHO-K1 cells, the antibody obtained by the method has higher yield, simple screening steps and higher biological activity.

Description

Method for preparing anti-IL-12 and/or IL-23 monoclonal antibody

Cross Reference to Related Applications

The present application claims priority of patent application No.202110174184.3 entitled "a method for preparing monoclonal antibodies against IL-12 and/or IL-23" filed on 9/2/2021 with the intellectual property office of china, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The invention relates to a method for preparing anti-IL-12 and/or IL-23 monoclonal antibody, in particular to a method for preparing Usunitumumab.

Background

Interleukins are a class of cytokines that are produced by and act on a wide variety of cells. Interleukin-12 (IL-12) is a cytokine with a wide range of biological activities, produced mainly by B cells and macrophages, and its molecule is a heterodimer, consisting of two subunits, p35 and p 40. The structure of interleukin-23 (IL-23) has homology with IL-12, exists in a heterodimer form, has an alpha chain of p19 and contains 189 amino acids, and a beta chain of the alpha chain is the same as p40 of IL-12. Its function is to activate stimulated T cells, promote their proliferation and production of interferon gamma, and induce proliferation of memory T cells. IL-12 and IL-23 play an important role in a series of processes such as maturation, activation, proliferation and immunoregulation of immune cells, and in addition, they are involved in various physiological and pathological reactions of the body.

As IL-12 and IL-23 are involved in various disorders, several therapeutic strategies have been devised to inhibit IL-12 and/or IL-23 activity. Among them, the drug "Ustekinumab" of Qiangsheng corporation was approved by the US FDA in 2009 and marketed, and is used for treating adult patients with moderate and severe plaque psoriasis. The Usunitumumab is an IgG1 antibody of a common subunit p40 of targeted IL-12 and IL-23, is a fully human monoclonal antibody, and can inhibit the biological activity of IL-12 and IL-23, thereby preventing the initial T cells from differentiating to Th1 and Th17 cells and further inhibiting the development of inflammatory response. Ubsunitumumab has been approved for the treatment of plaque psoriasis, psoriatic arthritis and crohn's disease in both the united states and europe, and ubsunitumumab has been approved for the treatment of moderate-severe plaque psoriasis and adult crohn's disease indications in china.

Usunuzumab of Qiangsheng corporation is an antibody expressed by SP2/0 cell, and the antibody expressed by mouse myeloma cell SP2/0 has low yield and strong immunogenicity (because of containing NGNA sialylation and alpha-1, 3 galactosylation). Therefore, there is a need to develop a new method for expressing antibodies, which further improves the yield and reduces the immunogenicity while ensuring the biological activity of the antibodies.

Disclosure of Invention

In a first aspect, the present invention provides a method of producing an anti-IL-12 and/or IL-23 monoclonal antibody or an antigen-binding fragment thereof, comprising expressing said monoclonal antibody or said antigen-binding fragment using a DHFR knockout or GS knockout CHO cell, said monoclonal antibody or said antigen-binding fragment comprising a heavy chain variable region comprising VH-CDR1, VH-CDR2 and VH-CDR3 having the amino acid sequences shown in SEQ ID No. 1, SEQ ID No.2 and SEQ ID No. 3, respectively; the light chain variable region comprises VL-CDR1, VL-CDR2 and VL-CDR3 which have the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

In one embodiment, the anti IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof contains SEQ ID NO. 7 heavy chain variable region and SEQ ID NO. 8 light chain variable region.

In one embodiment, the monoclonal antibody is an ubsunno monoclonal antibody.

Through a large number of experiments, the inventor unexpectedly finds that the monoclonal antibody expressed by DHFR gene knockout CHO cells or GS gene knockout CHO cells has unexpectedly higher biological activity and higher yield compared with antibodies expressed by SP2/0 cells and CHO-K1 cells.

In one embodiment, the invention provides a method of preparing an anti IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof, the method comprises the following steps:

a) cloning the coding sequence of the anti-IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof into an expression vector to obtain a recombinant expression vector, wherein the expression vector preferably contains a selection marker gene, and more preferably the selection marker gene is a DHFR gene or a GS gene;

b) transferring the recombinant expression vector obtained in the step a) into a CHO cell with a DHFR gene knockout or a CHO cell with a GS gene knockout to obtain a CHO cell modified by genetic engineering;

c) culturing the genetically engineered CHO cell obtained in step b), and selecting a monoclonal cell strain expressing the antibody;

d) culturing the monoclonal cell strain expressing the antibody obtained in the step c), harvesting a cell culture solution containing the antibody, and separating and purifying to obtain the monoclonal antibody.

In one embodiment, the method comprises the steps of:

a) cloning the coding sequence of the anti-IL-12 and/or IL-23 monoclonal antibody or the antigen binding fragment thereof into an expression vector to obtain a recombinant expression vector, wherein the expression vector preferably contains a selection marker gene, and more preferably the selection marker gene is a DHFR gene or a GS gene;

b) transferring the recombinant expression vector obtained in the step a) into a CHO cell with a DHFR gene knockout or a CHO cell with a GS gene knockout to obtain a CHO cell modified by genetic engineering;

c) culturing the genetically engineered CHO cell obtained in step b), and selecting a monoclonal cell strain which can efficiently express the antibody;

d) culturing the monoclonal cell strain which is obtained in the step c) and efficiently expresses the antibody, harvesting a cell culture solution containing the antibody, and separating and purifying to obtain the monoclonal antibody.

In one embodiment, the DHFR gene knockout CHO cell is a CHO-DG44 cell.

In one embodiment, the GS gene knockout CHO cell is obtained by knocking out the GS gene using Zinc Finger Nuclease (ZFN) technology or using CRISPR methods.

In one embodiment, the GS geneThe knockout CHO cell is

-a GS cell.

In one embodiment, the method comprises the steps of:

a) cloning the coding sequence of the anti-IL-12 and/or IL-23 monoclonal antibody or the binding fragment thereof into an expression vector to obtain a recombinant expression vector, wherein the expression vector contains a GS gene;

b) transferring the recombinant expression vector obtained in the step a) into

GS cells, genetically engineered

-a GS cell;

c) genetically engineering the gene obtained in step b)

-culturing GS cells in a glutamine-free medium and selecting a monoclonal cell line expressing said antibody;

d) culturing the monoclonal cell strain expressing the antibody obtained in the step c), harvesting a cell culture solution containing the antibody, and separating and purifying to obtain the monoclonal antibody.

In one embodiment, the cell culture process described in step d of the method is in fed-batch mode.

In a second aspect of the invention provides an anti IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof, through the first aspect of the method of the preparation.

In a third aspect, the invention provides a pharmaceutical composition, which comprises the monoclonal antibody prepared by the method of the first aspect, sucrose, L-histidine hydrochloride monohydrate and polysorbate 80.

In one embodiment, the pharmaceutical composition comprises 90 + -9 mg/mL of the monoclonal antibody prepared by the method of the first aspect of the invention, 76 + -7.6 mg/mL of sucrose, 0.5 + -0.3 mg/mL of L-histidine, 1 + -0.5 mg/mL of L-histidine hydrochloride monohydrate, and 800.04 + -0.004 mg/mL of polysorbate.

In one embodiment, the pharmaceutical composition comprises 90mg/mL of the monoclonal antibody prepared by the method of the first aspect of the invention, 76mg/mL of sucrose, 0.636mg/mL of L-histidine, 1.237mg/mL of L-histidine hydrochloride monohydrate, and 800.04mg/mL of polysorbate.

In a fourth aspect, the invention provides a pharmaceutical composition, which comprises the monoclonal antibody prepared by the method of the first aspect, L-histidine hydrochloride monohydrate, polysorbate 80, sucrose, disodium EDTA dihydrate and L-methionine.

In one embodiment, the pharmaceutical composition comprises 5 + -0.5 mg/mL of the monoclonal antibody prepared by the method of the first aspect of the invention, 0.77 + -0.077 mg/mL of L-histidine, 1.03 + -0.10 mg/mL of L-histidine hydrochloride monohydrate, 800.4 + -0.04 mg/mL of polysorbate, 85 + -8.5 mg/mL of sucrose, 0.02 + -0.002 mg/mL of EDTA disodium salt dihydrate and 0.4 + -0.04 mg/mL of L-methionine.

In one embodiment, the pharmaceutical composition comprises 5mg/mL of the monoclonal antibody prepared by the method of the first aspect of the invention, 0.77mg/mL of L-histidine, 1.03mg/mL of L-histidine hydrochloride monohydrate, 800.4 mg/mL of polysorbate, 85mg/mL of sucrose, 0.02mg/mL of disodium EDTA dihydrate, and 0.4mg/mL of L-methionine.

In a fifth aspect, the invention provides the method of the first aspect of the invention prepared by anti IL-12 and/or IL-23 monoclonal antibody or antigen binding fragments thereof or the third or fourth aspect of the invention of the pharmaceutical composition for the preparation of treatment of IL-12 and/or IL-23 associated diseases or disorders in the drug.

In one embodiment, the disease or disorder is selected from plaque psoriasis, psoriatic arthritis, crohn's disease, systemic lupus erythematosus, ulcerative colitis, multiple sclerosis, sarcoidosis, central spine arthritis, and ankylosing spondylitis. Preferably, the disease or disorder is selected from plaque psoriasis, psoriatic arthritis and crohn's disease.

The invention has the following beneficial effects:

1. the inventor unexpectedly finds that the monoclonal antibody produced by the method has the inhibiting activity on IL-12 and/or IL-23 which is more than 20 percent higher than that of the original medicine. By adopting the technical means of the invention, the inhibitory activity of the monoclonal antibody on IL-12 and/or IL-23 can be obviously improved.

2. The monoclonal antibody produced by the method has the yield as high as about 1.5 g/L.

Detailed Description

Interleukin-12 (also known as IL-12, cytotoxic lymphocyte maturation factor or natural killer cell stimulating factor) is a heterodimer composed of a p35 subunit and a p40 subunit, and many studies have shown that IL-12 plays a key role in promoting the Th1 immune response and in promoting host defense against intracellular pathogens. In addition, IL-12 may promote the development of several autoimmune and inflammatory diseases in driving a Th1 response. Interleukin-23 (IL-23) is a heterodimer consisting of the IL-12p40 subunit and its own p19 subunit, the receptor system of which shares the IL-12R β 1 subunit with IL-12R (. beta.1 and. beta.2), and the specific receptor subunit of which is IL-23R.

The biological effects of IL-12 and IL-23 differ from each other. IL-12 is secreted by activated inflammatory cells (monocytes, macrophages, neutrophils, microglia, dendritic cells). In the inflammatory process, IL-12 induces the production of interferon-gamma (IFN-. gamma.) by NK cells (natural killer cells) and T cells. IL-12 may then be combined with IFN-gamma to induce differentiation of T cells into Th1 cells. This response stimulates the cellular immune system and maximizes the killing effect of macrophages on pathogens and CD8⁺Proliferation of T cells (Trinchieri 2003Nat Rev Immunol 3133-46). Overproduction of IL-12 is associated with increased pro-inflammatory activity and tissue damage typical of autoimmunity (Leonard et al, 1997Crit Rev Immunol 17545-53). Dysregulation of IL-12 production is implicated in the following diseases: psoriasis (Yawalkar et al, 1998J Invest Dermatol 1111053-7), Crohn's disease (Neurath et al, 1995J Exp Med 1821281-90; simpson et al A,1998J Exp Med 1871225-34; camoglio et al, 2002Eur J Immunol 32261-9), multiple sclerosis (fasbender et al, 1998Neurology 51753-8; laman et al, 1998J neuro-immumunal 8630-45), rheumatoid arthritis (Kim et al, 2000 Clin Exp Immunol 119175-81), and other autoimmune diseases.

Since IL-12 and IL-23 contain common subunits, it is difficult to attribute a disease state to the overproduction of one interleukin or the other alone. However, studies have shown that dysregulation of IL-23 is involved in the following diseases: psoriasis (Lee et al, 2004J Exp Med 199125-30), Crohn's disease (Neurath 2007Nat Med 1326-8) and multiple sclerosis (Cua et al, 2003Nature 421744-8), among other autoimmune diseases.

The invention of the "anti IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof" refers to the ability to specific binding IL-12 and/or IL-23 antibody or antigen binding fragment thereof, preferably, anti IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof "binding IL-12 and/or IL-23, resulting in IL-12 and/or IL-23 biological activity inhibition; more preferably, anti IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof is anti IL-12 and IL-23 monoclonal antibody or antigen binding fragment thereof, causing IL-12 and/or IL-23 biological activity inhibition; more preferably, the heavy chain variable region of the monoclonal antibody comprises: VH-CDR1, VH-CDR2 and VH-CDR3 having the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO.2 and SEQ ID NO. 3, respectively; the light chain variable region of the monoclonal antibody comprises: VL-CDR1, VL-CDR2 and VL-CDR3 having amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively; more preferably, the monoclonal antibody comprises the heavy chain variable region shown in SEQ ID NO. 7 and the light chain variable region shown in SEQ ID NO. 8; most preferably, the monoclonal antibody is ubsunumab. As used herein, the term "inhibit" refers to a decrease in the biological activity of a cytokine when an antibody specifically binds to the cytokine. Inhibition of the biological activity of IL-12 and/or IL-23 by an antibody can be assessed by measuring one or more indicators of the biological activity of IL-12 and/or IL-23, which are well known in the art.

The term "Ustunzumab" refers to a product having the trade name Ustunzumab

The fully human IgG1 kappa antibody of (1), which binds to the p40 subunit of IL-12 and IL-23, thereby blocking the inflammatory response in the patient. The heavy chain and light chain sequences of the ubsunumab are as set forth in SEQ ID NO: 9 and SEQ ID NO: shown at 10. On the market, omeprazole has obtained approval for the treatment of moderate to severe plaque psoriasis, active psoriasis, and moderate to severe active crohn's disease. Ulvacizumab in China has been approved for the treatment of moderate to severe plaque psoriasis and adult Crohn's disease indications

In the present invention, the term "standard" is used to indicate that a comparison is made with a biologically similar product, a standard being a product that has gained market acceptance. In the case where the antibody prepared is ubsunumab, the standard is approved

And (5) producing the product.

The term "CHO cell" as used herein, derived from epithelial cells of the ovary of Chinese hamster (Tijo and Puck (1958) J. exp. Med.108: 259-271); the term "genetically engineered CHO cells" refers to CHO cells that have been modified or altered by any suitable genetic means and methods known to those skilled in the art such that they express a particular protein. Preferably, the genetically engineered CHO cell may express the monoclonal antibody or antigen binding fragment thereof. More preferably, the genetically engineered CHO cell is obtained by transfecting a recombinant expression vector containing a coding sequence of the monoclonal antibody or the antigen binding fragment thereof into a corresponding CHO cell line. Preferably, the CHO cell is a dihydrofolate reductase (DHFR) gene knockout or a Glutamine Synthetase (GS) gene knockout CHO cell; preferably, the DHFR gene knockout CHO cell is a CHO-DG44 cell. Preferably, the GS gene knockout CHO cell knocks out GS by using Zinc Finger Nuclease (ZFN) technology or using CRISPR methodA gene is thus obtained; more preferably, the GS gene knockout CHO cell is

-a GS cell.

As used herein, "CHO-DG 44 cells" refers to CHO cells in which the biallelic dihydrofolate reductase (DHFR) gene has been knocked out, and is a mammalian cell expression system developed by Thermo-Fisher that is currently widely used for recombinant protein expression. Herein, GS Gene knockout CHO cell such as one that knocks out the GS Gene using Zinc Finger Nuclease (ZFN) technology or using CRISPR method, for example, Merck's CHO cell

GS mammalian cell expression System, using

Endogenous Glutamine Synthetase (GS) genes of CHO-K1 cell strains are knocked out by a Zinc Finger Nuclease (ZFN) technology, so that the cell growth process must depend on the addition of exogenous L-glutamine to form a natural screening marker, and the dependence of a GS screening system on L-methionine sulfone imine (MSX) is also eliminated. The plasmid is not transferred into the culture medium with glutamine, but the plasmid (with GS gene) can survive in the culture medium without glutamine, so that the high expression cell strain is screened without adding MSX and other resistance screening matter, and this simplifies the screening process and makes the process suitable for industrial production.

The inventors have unexpectedly found, through a large number of experiments, that CHO cells or GS (glutamine synthetase) cells, in particular CHO-DG44 cells or CHO-DG44 cells, which were knocked out using DHFR (dihydrofolate reductase) gene

GS cells expressing said monoclonal antibody, with higher biological activity, higher yield, lower immunogenicity and higher cell stability than those expressed by SP2/0 cells and CHO-K1 cells, in particular UsunuzumabGood, the screening step is simple.

The method for preparing the monoclonal antibody comprises the following steps:

a) cloning the coding sequence of the monoclonal antibody into an expression vector to obtain a recombinant expression vector, wherein the expression vector preferably contains a selection marker gene, and more preferably the selection marker gene is a DHFR gene or a GS gene;

b) transferring the recombinant expression vector obtained in the step a) into a CHO cell with a DHFR gene knockout or a CHO cell with a GS gene knockout to obtain a CHO cell modified by genetic engineering;

c) culturing the genetically engineered CHO cell obtained in step b), and selecting a monoclonal cell strain expressing the antibody;

d) culturing the monoclonal cell strain expressing the antibody obtained in the step c), harvesting a cell culture solution containing the antibody, and separating and purifying to obtain the monoclonal antibody.

Methods for constructing recombinant expression vectors suitable for expressing antibodies in CHO cells are well known to those skilled in the art, for example, in Makrides et al (1999) Protein Expr. Purif.17:183-202 and Kaufman (2000) mol. Biotechnol.16: 151-161. The expression vector refers to a vector, such as a recombinant plasmid, which can express a target gene by adding expression elements (such as a promoter, a terminator and the like) on the basis of a basic skeleton of a cloning vector. Preferably, the expression vector contains a selectable marker gene. The selectable marker gene encodes a protein as described below: (a) conferring resistance to antibiotics or other toxins, (b) supplementing auxotrophic deficiencies, or (c) providing key nutrients not available from complex media. More preferably, the selectable marker gene is a dihydrofolate reductase gene or a glutamine synthetase gene. Methods for genetically engineering CHO cells are well known to those skilled in the art and specifically include co-transfecting CHO cells with one expression vector containing genes encoding the heavy and light chains of an antibody or a first expression vector containing genes encoding the heavy chain of an antibody and a second expression vector containing genes encoding the light chain of an antibody. Will recombineMethods for transferring expression vectors into CHO cells include electroporation, calcium phosphate precipitation, lipofection, DEAE-dextran transfection, and the use of commercial transfection kits, such as FreeStyle from Thermo fisher Scientific^TMMAX Reagent, etc.

Culturing the obtained genetically engineered CHO cells in a culture medium to obtain a monoclonal cell strain capable of efficiently expressing the antibody. Wherein, for CHO cells with DHFR (dihydrofolate reductase) gene knockout, corresponding inhibitors such as Methotrexate (MTX) and the like are added into a target culture medium according to a selection marker gene carried on a transfected recombinant expression vector,

And the like, thereby obtaining a cell line capable of efficiently expressing the antibody; for the CHO cell with GS (glutamine synthetase) gene knockout, because the cell can survive in the culture medium with glutamine when the plasmid is not transferred and can survive in the culture medium without glutamine when the plasmid is transferred (the GS gene is on the plasmid), the cell strain which can express the antibody with high efficiency can be obtained by culturing in the culture medium without glutamine, Methionine Sulphonamide (MSX) is not added in the culture medium,

and the like. Then carrying out monoclonal screening to obtain a monoclonal cell strain expressing the antibody; preferably, a monoclonal cell strain capable of expressing the antibody with high efficiency is obtained.

The obtained genetically engineered CHO cells were cultured in an appropriate medium to produce antibodies. "culture medium" as described herein, refers to a solution containing nutrients required for mammalian cell growth. In general, the culture medium provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cells.

There are different culture modes for genetically engineered CHO cells, including batch, continuous and fed-batch. In the present invention, it is preferable to produce an antibody using a fed-batch culture mode. In fed-batch culture, the culture is started with a certain amount of medium, and during the subsequent culture, one or more nutrients are supplemented in order to prevent the depletion of nutrients, during which no product is removed from the cell culture medium.

After antibody production by genetically engineered CHO cells, since recombinant proteins expressed from mammalian cells, particularly antibodies, are usually secreted into the cell culture broth during the culture process, the antibodies are harvested by separating the cell culture broth containing the antibodies from the cells at the end of the culture process. Cell separation methods should minimize cell lysis, avoid the increase in cell debris, and release proteases and other molecules that may affect the quality of the immunoglobulin product. The cell culture fluid containing the antibody is typically obtained by means of centrifugation and/or filtration, the recombinant protein being present in the supernatant or filtrate, respectively.

After obtaining the cell culture fluid containing the antibody, the antibody is purified from the cell culture fluid. Purification of recombinant proteins is typically accomplished by a series of chromatographic steps, such as anion exchange chromatography, cation exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and the like. Purification of recombinant antibodies typically begins with ProteinA affinity chromatography followed by one or more additional chromatography steps, such as cation exchange chromatography. Further, the purification step may include ultrafiltration, diafiltration, and the like.

As described above, the inventors found that the ubsumab antibody standard substance produced by the method of the present invention

The product has higher bioactivity. The biological activity of Ustuzumab is expressed by the ability to bind to IL-23, the ability to bind to IL-12, the ability to bind to FcRn and/or the inhibition of IL-12 and/or IL-23 induced gene expression, in particular the inhibition of IL-12 and/or IL-23 induced gene expression. The ability of ubsumab to bind to IL-23, IL-12 or FcRn can be assessed by, but is not limited to, ELISA and biolayer interferometry; gene table induced by Usunitumumab on IL-12Inhibition of expression can be studied by assaying samples for cells that are reactive to IL-12 (e.g., T lymphocytes), IL-12 and antibodies, and then detecting the production of a target molecule, such as interferon-gamma (IFN-gamma). The target molecule interferon-gamma (IFN-gamma) may be detected and/or quantified by any suitable method, such as ELISA.

The inhibitory action of the Usneuzumab produced by the method on the generation of IFN-gamma is larger than that of a standard product on the generation of IFN-gamma, and the inhibitory action of the Usneuzumab produced by the method on the generation of IFN-gamma exceeds that of the standard product on the generation of IFN-gamma by more than 20%.

The invention also relates to a pharmaceutical composition containing the anti-IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof prepared by the method. In addition to comprising the monoclonal antibody, the pharmaceutical composition of the invention further comprises at least one pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are substances which do not affect the physiological activity of the active substance, such as ubenizumab, and which are capable of stabilizing the pharmaceutical ingredients and/or increasing the solubility or reducing the viscosity of the pharmaceutical composition. Common pharmaceutical excipients for monoclonal antibodies include buffers, salts, sugars or sugar alcohols, amino acids and surfactants.

In one embodiment, a pharmaceutical composition comprises the monoclonal antibody, sucrose, L-histidine hydrochloride monohydrate, and polysorbate 80; preferably, the pharmaceutical composition comprises the monoclonal antibody 90 + -9 mg/mL, sucrose 76 + -7.6 mg/mL, L-histidine 0.5 + -0.3 mg/mL, L-histidine hydrochloride monohydrate 1 + -0.5 mg/mL, and polysorbate 800.04 + -0.004 mg/mL. More preferably, the pharmaceutical composition comprises the monoclonal antibody 90mg/mL, sucrose 76mg/mL, L-histidine 0.636mg/mL, L-histidine hydrochloride monohydrate 1.237mg/mL, and polysorbate 800.04 mg/mL.

In another embodiment, a pharmaceutical composition comprises the monoclonal antibody, sucrose, L-histidine hydrochloride monohydrate, EDTA disodium salt dihydrate, L-methionine and polysorbate 80. Preferably, the pharmaceutical composition comprises: the monoclonal antibody is 5 +/-0.5 mg/mL, the L-histidine is 0.77 +/-0.077 mg/mL, the L-histidine hydrochloride monohydrate is 1.03 +/-0.10 mg/mL, the polysorbate is 800.4 +/-0.04 mg/mL, the sucrose is 85 +/-8.5 mg/mL, the disodium EDTA dihydrate is 0.02 +/-0.002 mg/mL, and the L-methionine is 0.4 +/-0.04 mg/mL. More preferably, the pharmaceutical composition comprises: the monoclonal antibody is 5mg/mL, the L-histidine is 0.77mg/mL, the L-histidine hydrochloride monohydrate is 1.03mg/mL, the polysorbate is 800.4 mg/mL, the sucrose is 85mg/mL, the disodium EDTA dihydrate is 0.02mg/mL, and the L-methionine is 0.4 mg/mL.

The anti-IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof or the pharmaceutical composition can be used for preparing a medicament for treating IL-12 and/or IL-23 related diseases or disorders; preferably, the disease or disorder is selected from plaque psoriasis, psoriatic arthritis, systemic lupus erythematosus, crohn's disease, ulcerative colitis, multiple sclerosis, sarcoidosis, central spine arthritis, and ankylosing spondylitis. More preferably, the disease or disorder is selected from plaque psoriasis, psoriatic arthritis and crohn's disease.

In one embodiment, the anti-IL-12 and/or IL-23 monoclonal antibodies or antigen-binding fragments thereof or the pharmaceutical compositions described herein are useful for treating plaque psoriasis, particularly in treating adults with moderate to severe plaque psoriasis, patients who are unresponsive or intolerant to other systemic therapies, including cyclosporine, Methotrexate (MTX) or PUVA (psoralen and ultraviolet a), and in treating patients who are 12 years old and older, in adolescent to severe plaque psoriasis, who are inadequately controlled or intolerant to other systemic therapies or phototherapy therapies, and in treating psoriatic arthritis, particularly in treating active psoriasis, in adult patients who have been inadequately treated with previously non-biological disease modifying antirheumatic drugs (DMARDs).

In another embodiment the anti-IL-12 and/or IL-23 monoclonal antibodies or antigen-binding fragments thereof or the pharmaceutical compositions of the invention are useful for the treatment of Crohn's disease, in particular adult patients with moderate to severe active Crohn's disease, who are poorly responsive, unresponsive, intolerant or medically contraindicated to conventional therapies or TNF-a antagonists.

A composition or method described herein as "comprising" or "including" one or more named elements or steps is open-ended, meaning that the named elements or steps are necessary, but that other elements or steps may be added within the scope of the composition or method. Known or disclosed equivalents of any named base element or step may be substituted for the recited element or step in any composition or method disclosed herein.

Detailed Description

Example 1 construction of CHO-DG44 cell line expressing Usunitumumab

Vector construction

(1) Preparation of antibody heavy chain protein expression vector

Two ends of a coding sequence (SEQ ID No:11) of the Usunuzumab heavy chain are designed with two enzyme cutting sites of BamHI and Xhol, which are respectively connected with the BamHI and Xhol enzyme cutting sites on an expression vector pCDNA3.1 (purchased from Thermo Fisher Scientific company) to realize the connection of a target protein coding DNA fragment and the expression vector and obtain recombinant plasmid DNA. The recombinant plasmid DNA is introduced into Escherichia coli DH5 a (purchased from Tiangen Biochemical technology Co., Ltd.), single colony plasmid is extracted and sent for sequencing, and plasmid with correct sequencing result is obtained and is marked as recombinant plasmid pCDNA-HC.

(2) Preparation of antibody light chain protein expression vector

HindIII and Xhol restriction sites are designed at two ends of a coding sequence (SEQ ID No:12) of a Ustunzumab light chain and are respectively connected with HindIII and Xhol restriction sites on an expression vector pOptiVEC (purchased from Thermo Fisher Scientific company), so that a target protein coding DNA fragment is connected with the expression vector to obtain recombinant plasmid DNA, the recombinant plasmid DNA is introduced into escherichia coli DH5 a (purchased from Tiangen Biotechnology Co., Ltd.), a single colony plasmid is extracted and sent to be sequenced to obtain a plasmid with a correct sequencing result, and the plasmid is marked as recombinant plasmid pOptiVec-LC.

Construction of CHO-DG44 cell strain for expressing Usunitumumab

The recombinant plasmids pOptiVec-LC and pCDNA-HC were linearized, transfected into CHO-DG44 cells (purchased from Thermo Fisher Scientific Co.) and cultured to obtain a cell line highly expressing the target protein. The method comprises the following specific steps:

(1) recombinant plasmid transfected CHO-DG44 cells

The plasmid was linearized using PvuI enzymatic cleavage sites on recombinant plasmids pOptiVec-LC and pCDNA-HC. The enzyme-digested plasmid DNA was concentrated to a concentration of 1ug/ul by ethanol precipitation.

Take 1.5X 10⁷The CHO-DG44 cells were replaced with 30mL of CD DG44 medium (purchased from Thermo Fisher Scientific Co.) preheated to 37 ℃ and mixed well; 60ug of plasmid DNA (pcDNA-HC to pOptiVec-LC molar ratio 1:1) and 15uL FreeStyle^TMMAX Reagent (available from Thermo Fisher Scientific Co.) was placed in 1.2mL OptiPRO^TMSFM medium (purchased from Thermo Fisher Scientific Co.) was gently mixed and incubated at room temperature for 10min to obtain DNA-Reagent complex. 1.2mL of the DNA-Reagent complex was added to 125mL of the CHO-DG44 cell shake flask, the flask was gently shaken, and the flask was placed in a shaker at 37 ℃ with 8% CO₂The rotation speed was 130-. 48 hours after transfection, cells were passaged to hypoxanthine/thymine (HT) -deficient CD OptiCHO^TMComplete medium (purchased from Gibco Life Technologies, Inc.).

(2) Selection of stably transfected cells

First wheel

Screening: counting the cell lines obtained in the last step; in a preheated medium containing 500ug/mL

CD OptiCHO of reagent (available from Thermo Fisher Scientific Co.)^TMCells were diluted in complete medium (purchased from Gibco Life Technologies) to a final cell density of 5X 10⁵Viable cells/mL; the flask was placed in an incubator at 37 ℃ with 8% CO₂Under the condition of humidified air, the rotating speed is 130-. Replacing appropriate amount of fresh culture medium every 2-3 days, and maintaining for 14-21 days untilCell survival rate>90％。

Second round MTX gradient screen: the first round of screened cells were counted, centrifuged at 1000rpm for 5 minutes, the old medium was discarded, and the cells were seeded into CD OptiCHO containing 50nM MTX^TMComplete medium (purchased from Gibco Life Technologies, Inc.; containing 500. mu.g/mL)

) Inoculation density of 3X 10⁵cells/mL. The flask was placed in an incubator at 37 ℃ with 8% CO₂Under the condition of humidified air, the rotating speed is 130-. Changing an appropriate amount of fresh medium every 2-3 days, inoculating cells to CD OptiCHO containing 100nM MTX at a cell viability rate greater than 80%^TMComplete medium (purchased from Gibco Life Technologies, Inc.; containing 500. mu.g/mL)

) Inoculation density of 3X 10⁵cells/mL, the flasks were placed in an incubator at 37 ℃ with 8% CO₂Under the condition of humidified air, the rotating speed is 130-. Changing an appropriate amount of fresh culture medium every 2-3 days, inoculating cells to CD OptiCHO containing 500nM MTX at a cell viability rate of more than 80%^TMComplete medium (purchased from Gibco Life Technologies, Inc.; containing 500. mu.g/mL)

) Inoculation density of 3X 10⁵cells/mL, total incubation time 20-30 days, until cell viability>90％。

(3) Monoclonal screening and expanded culture

The CELLs obtained in the above step were diluted with a Cloning Medium EX-CELL CHO Cloning Medium (purchased from Sigma Aldrich) in a gradient manner to 1000 viable CELLs/mL, and seeded into a 96-well plate at 0.8 CELLs per well. The inoculated 96-well plate was placed at 37 ℃ with 5% CO₂The plate was incubated still in the incubator under the air condition for 10 to 14 days, 11 days in this example, and the wells were examined using a microscopeGrowth of single clone colonies.

When the cells in a 96-well plate grow to reach 80% confluence, the yield of each clone is detected by an ELISA method, specifically, the content of the ubsunitumumab in the fermentation liquor is determined by a double-antibody sandwich method, the coating antibody is a murine monoclonal antibody human IgG Fc fragment antibody (purchased from Sigma under the product number I6260-5 mL), the secondary antibody is a murine monoclonal antibody human IgG Fab fragment antibody (purchased from Kingsler Biotech Co., Ltd under the product number A01855), and the used standard product is original research medicine

The maximum dilution concentration is 1ug/mL, and 12 standards are diluted in a twofold gradient.

The first 25% of clones with high screening yields were clonally expanded, and desired monoclonal cells were aseptically collected and expanded into 24-well plates. After 3-5 days, the first 10% of the higher yield clones were selected and transferred to 6-well plates using the same assay and cultured. Using the same assay, monoclonal cells were selected for the first 10% yield in 6-well plates and each cell line was transferred individually to T-25 flasks for expansion when the number of cells in the T-25 flask reached 1x10⁷Thereafter, the cells were transferred to 125mL culture flasks. At 37 ℃ and with 8% CO₂Under the air condition of (1), the oscillation speed is 130-135rpm, the fed-batch culture mode is adopted for culture, and a fed-batch culture medium Dynamis is added in the culture process^TMMedium (purchased from Thermo Fisher company) cultured for 14 days, and collected cell fermentation broth of CHO-DG44 cell strain highly expressing ubeniumumab.

Example 2 expression of Usunitumumab

Construction of GS cell line

Vector construction

(1) Expression of antibody heavy chain protein and vector ligation

Two restriction sites BstB I and Pac I are designed at two ends of a coding sequence of the Usunu monoclonal antibody heavy chain and are respectively connected with the restriction sites BstB I and Pac I on an expression vector pCGS (purchased from Merck KGaA company), so that the connection of a target protein coding DNA fragment and the expression vector is realized, and the recombinant plasmid DNA is obtained. The recombinant plasmid DNA was introduced into E.coli DH5 a (purchased from Tiangen Biochemical technology Co., Ltd.) and applied to LB agar medium (prepared per liter of medium, 950ml of deionized water to which 10g of tryptone, 5g of yeast extract, 10g of NaCl10g, and then deionized water to 1L) containing 100ug/ml of ampicillin (final concentration). The obtained clone was cultured in LB liquid medium containing 100ug/ml ampicillin (final concentration), and plasmids were extracted with a plasmid extraction kit (purchased from Tiangen Biochemical technology Co., Ltd.), identified by nucleic acid sequencing, and plasmids with correct sequencing results were selected and designated as recombinant plasmids pCGS-HC.

(2) The light chain protein of the expression antibody is connected with the carrier

HindIII and Xhol restriction sites are designed at two ends of a coding sequence of the Usunu monoclonal antibody light chain and are respectively connected with HindIII and Xhol restriction sites on the recombinant plasmid pCGS-HC, so that the connection of a target protein coding DNA fragment and an expression vector is realized, and new recombinant plasmid DNA is obtained. The recombinant plasmid DNA was introduced into E.coli DH5 a (purchased from Tiangen Biochemical technology Co., Ltd.) and applied to LB agar medium (prepared per liter of medium, 950ml of deionized water to which 10g of tryptone, 5g of yeast extract, 10g of NaCl, and then 1L of deionized water) containing 100ug/ml of ampicillin (final concentration). The obtained clone was cultured in LB liquid medium containing 100ug/ml ampicillin (final concentration), and plasmids were extracted with a plasmid extraction kit (purchased from Tiangen Biochemical technology Co., Ltd.), identified by nucleic acid sequencing, and plasmids with correct sequencing results were selected and designated as recombinant plasmids pCGS-HC-LC.

Cell line selection

Transfection of recombinant plasmid pCGS-HC-LC into

GS cells (purchased from Merck KGaA) were screened to obtain cell lines highly expressing the target protein. The method comprises the following specific steps:

(1) transfection of recombinant plasmids

-GS cell

a. Cells were passaged the day before transfection to a density of 1X10 at transfection⁶Viable cells/mL.

b. Cell count, total number of cells required per sample 6X 10⁶The desired cells were centrifuged at 1000rpm for 10min in a 50mL centrifuge tube.

c. Each sample was resuspended in 600. mu.L LEX-CELL CD CHO medium (purchased from Sigma Aldrich) and 40. mu.g of plasmid (plasmid concentration > 1. mu.g/. mu.L) was added and mixed well, and then placed in a refrigerator together with an electric rotor for precooling at 4 ℃.

d. The CELLs added with the plasmid are blown uniformly and then added into an electric rotating cup, the voltage is 300V for electric rotation, after the electric rotation is finished, the CELLs are added into a culture medium EX-CELL CD CHO Fusion (purchased from Sigma Aldrich company) containing 6mM glutamine and preheated at 37 ℃, and the CELLs are placed into a carbon dioxide incubator for static culture for 48 hours.

(2) Selection of stably transfected cells

After 48h, the CELLs were passaged to glutamine-free medium EX-CELL CD CHO Fusion (from Sigma Aldrich). Passage was performed every 2 days, with each passage labeled "P1", "P2", where n is the passage of the cell. Before each passage, 40. mu.L of the cell suspension was subjected to cell counting and cell viability analysis using a Countstar IC 1000 automatic cell counter to determine the viable cell density and viability of each generation of cells. When the cell viability was > 95%, the screening process was completed. The process can also be completed by a method of screening a cell pool (minipool).

(3) Monoclonal screening and expanded culture

The cells obtained in the previous step were seeded into 96-well plates, 0.5-1 cells per well.

a. 400mL of complete Cloning Medium EX-CELL CHO Cloning Medium (available from Sigma Aldrich) was preheated to 37 ℃;

b. counting the cells to be plated, and calculating the number of the required cells, namely 20 96-well plates, wherein 960 cells are required for calculation of 0.5 cell per well;

c. continuously diluting cells to a final concentration of 1000 viable cells/mL by using the complete cloning medium, and taking 960uL of cell diluent to a preheated cloning medium;

d. gently mixing the cell suspension, and transferring the cell suspension into a sterile sample adding groove; using a multichannel pipettor, dispense 200 μ Ι _ of the diluted cells into a 96-well plate under sterile conditions;

e. at 37 deg.C, 5% CO₂The plate was incubated still in the incubator under air conditions for 10 to 14 days, 11 days in this example, and the growth of the monoclonal colonies in the wells was examined using a microscope.

When the CELLs in the 96-well plate were grown to 80% confluence, the yields of the individual clones were examined by the same ELISA method as in example 1, the first 25% clones with high productivity were selected for clonal expansion, the desired clones were aseptically collected, gently pipetted up and down and the entire contents of each well were transferred to each well of a 24-well plate containing 1mL of fresh medium EX-CELL CD CHO Fusion (available from Sigma Aldrich).

After 3-5 days, using the same assay, the first 10% higher yield clones of interest were selected and transferred to 6-well plates, the desired clones were collected aseptically, and fresh medium EX-CELL CD CHO Fusion was added to each well of the 6-well plates, and finally the culture volume of each well was 3 mL.

Using the same assay, the first 10% yield of monoclonal cell lines in 6-well plates were selected and each cell line was transferred individually to T-25 flasks for expansion when the number of cells in the T-25 flask reached 1x10⁷Thereafter, the cells were transferred to 125mL culture flasks. At 37 ℃ and with 8% CO₂Under the air condition of (1), the oscillation speed is 130-135rpm, the fed-batch culture mode is adopted for culture,

during the culture process, a Feed medium EX-CELL Advanced CHO Feed 1 (purchased from Sigma Aldrich company) is added, and after 14 days of culture, the high-expression ubsunobumab is collected

-cell fermentation broth of GS cell line.

Example 3 antibody purification procedure

High table to be collectedCHO-DG44 cell strain of dacustitumumab and

-cell fermentation broth of GS cell line, subjected to the following operations:

1. centrifugation

Setting the rotating speed of a centrifuge to 3000rpm, centrifuging for 10min, and removing cells; the centrifuge was then set at 15000rpm for 20min to remove cell debris. The step can also adopt a deep filtration method to clarify the cell fermentation liquor.

2. Microfiltration

The samples were microfiltered using a Perl sterile grade filter (pore size 0.22 μm, material polyethersulfone).

3. Elution is carried out

The instrument comprises the following steps: AKTA protein purification apparatus and Mabselect sure LX packing (both from GE Co.)

Reagent: balance liquid: 150mM NaCl, 20mM phosphate buffer pH7.4

Eluent 1: 1M NaCl 20mM sodium citrate pH5.5

Eluent 2: 20mM sodium citrate pH5.5

Eluent: 20mM sodium citrate pH3.0

Regeneration liquid: 0.1M NaOH

The method comprises the following steps: 1) balancing by using the balancing liquid; 2) sampling; 3) leaching: sequentially leaching with leacheate 2, leacheate 1 and leacheate 2; 4) eluting with the eluent; 5) clean In Place (CIP) using 0.5M sodium hydroxide; 6) rebalancing: the above-mentioned equilibrium liquid was used for equilibration.

The Usunuzumab product with the purity of 97% is finally obtained by using the CHO-DG44 cell strain to produce the Usunuzumab, and the yield is about 1.5 g/L.

Use of

And producing the Usunuzumab by using the cells of the GS cell strain to finally obtain the Usunuzumab product with the purity of 98 percent, wherein the yield is about 1.5 g/L.

Example 4 Usunitumumab expressed by CHO-DG44 cells had inhibitory Activity on IL-12

Isolation and activation of T cells

Peripheral Blood Mononuclear Cells (PBMC) from Redbold were sorted for total CD3 by removing non-T cells with magnetic bead sorting reagent (STEM Cell) according to the EasySep Human T Cell Isolation Kit protocol⁺T cells. T cells at a concentration of 1x10⁶The culture was carried out in 12-well plates in 1640 medium (from Sigma Aldrich) containing 10% FBS (fetal bovine serum) (Gibco Life Technologies) or in TexMACX medium (from Miltenyi Biotec) in the absence of serum, and in the case of T-cells, the stimulator anti-CD3/CD28 antibody (from Thermo Fisher Scientific) was added together with IL-2(30u/mL) (from Thermo Fisher Scientific).

Mixing 1.0X 10⁶ml^-1T cells were pre-stimulated with magnetic beads to produce interferon-gamma (IFN-gamma), and 2 days later, the cell concentration was adjusted to 0.5X 10 by centrifugation of the medium on day 3⁶ml^-1(exchange liquid into TexMACX medium).

IL-12 ELISA

The strips required for the assay were removed from the sealed bags equilibrated to room temperature using a human IFN-. gamma.ELISA kit (purchased from Shanghai Eikes Co.) leaving blank wells. Regulating the T cells obtained in the previous step to 0.5X 10⁵IL-12, the inventive Usunuzumab sample (hereinafter, referred to as "sample") and a standard were added to the corresponding wells (100 ul/well) in the amounts shown in Table 1, respectively, and cell activity experiments were performed according to the design of Table 1. The reaction wells were sealed with a sealing plate of gummed paper and incubated at 37 ℃ for 90 minutes in an incubator. Plates were washed 5 times and biotinylated antibody working solution (100 ul/well) was added except for blank wells. The reaction wells were sealed with a sealing plate of gummed paper and incubated at 37 ℃ for 60 minutes in an incubator. The plate was washed 5 times and the enzyme conjugate working solution (100 ul/well) was added except for blank wells. The reaction wells were sealed with a sealing plate gummed paper, incubated at 37 ℃ for 30 minutes in the dark. The plate was washed 5 times, 100ul of chromogenic substrate (including blank wells) was added per well, incubated at 37 ℃ for 15 minutes in the absence of light. Add stop solution (including blank wells) 100 ul/well and measure OD450nm immediately after mixing.

TABLE 1 cell Activity experiments and results of Usunuzumab samples

Inhibition rate (Abs)_{(IL-12 average value)}–Abs_(antibodies))/(Abs_{(IL-12 average value)}–Abs_{(average value of cell supernatant)})

According to the data results and the inhibition rate calculation formula in table 1, the inhibition rate of the Usunitumumab sample expressed by CHO-DG44 on the IL-12 activity is 43.19%, the inhibition rate of the standard product on the IL-12 activity is 34.9%, and the inhibition activity of the Usunitumumab sample expressed by CHO-DG44 on the IL-12 is 23.753% higher than that of the Usunitumumab standard product.

Example 5

Inhibitory Activity of Usunitumumab expressed in GS cell against IL-12

Isolation and activation of T cells

Peripheral Blood Mononuclear Cells (PBMC) from Redbold were sorted for total CD3 by removing non-T cells with magnetic bead sorting reagent (STEM Cell) according to the EasySep Human T Cell Isolation Kit protocol⁺T cells. T cells at a concentration of 1x10⁶The cells were cultured in 12-well plates in X-vivo (available from Lonza) and, in the case of T-cell culture, the stimulator anti-CD3/CD28 antibody (available from Thermo Fisher Scientific Co.) was added together with IL-2(30u/mL) (available from Thermo Fisher Scientific Co.) added.

Regulatory T cell concentration of 1X10⁶one/mL. Mixing 1.0X 10⁶ml^-1T cells were pre-stimulated with magnetic beads to activate them and produce gamma interferon (gamma-IFN), and 2 days after stimulation, the cell concentration was adjusted to 0.3X 10 by centrifugation on day 3⁶ml^-1。

IL-12ELISA

This step uses a human IFN-. gamma.ELISA kit (purchased from Shanghai Eikes Co.), removes the 96-well plate of the plate required for the assay from the sealed bag equilibrated to room temperature, and presses IL-12Adding the amount shown in table 2 into a 96-well plate (50 uL/well), adding the inventive ubepratumab sample (hereinafter referred to as "sample") and the standard sample into the corresponding well (50 uL/well) according to the amount shown in table 2, incubating in an incubator for 1h, centrifuging the T cells obtained in the previous step to remove the original culture medium, and adjusting the T cells to 0.3 × 10 with X-vivo⁵Per well, and then added to the corresponding well (100 uL/well). The reaction wells were sealed with a sealing plate of gummed paper and incubated at 37 ℃ for 90 minutes in an incubator. The plate was washed 5 times and biotinylated antibody working solution (100 ul/well) was added. The reaction wells were sealed with a sealing plate of gummed paper and incubated at 37 ℃ for 60 minutes in an incubator. The plate was washed 5 times and the enzyme conjugate working solution (100 ul/well) was added. The reaction wells were sealed with a sealing plate gummed paper, incubated at 37 ℃ for 30 minutes in the dark. Washing the plate for 5 times, adding 100ul of chromogenic substrate per hole, incubating in an incubator at 37 ℃ and keeping dark for 15 minutes. Add stop solution 100 ul/well and mix well immediately before measuring OD450nm value.

TABLE 2 cellular Activity experiments and results of Usunuzumab samples

From the data in table 2, the inhibition ratio of each antibody as in table 3 was calculated using the following inhibition ratio calculation formula.

Inhibition rate (Abs)_(IL-12)–Abs_{(Standard article)})/(Abs_(IL-12)–Abs_{(cell supernatant)})

TABLE 3 Experimental results treatment

Antibodies	0.5ug of standard	0.5ug sample	1ug of standard article	1ug sample
					Inhibition ratio (%)	9.52	11.67	27.64	37.98

It can be calculated that when 0.5ug of Usunitumumab was used in the above experiment,

the USTRINOMAB sample expressed in GS had an IL-12 inhibitory activity 22.58% higher than that of USTRINOMAB standard, and when 1ug USTRINOMAB was used in the above experiment,

the Usunitumumab sample expressed by GS has 37.41% higher IL-12 inhibition activity than the Usunitumumab standard product.

Through the experimental results of example 4 and example 5, it can be seen that the inhibitory activity of the ubsumizumab sample of the present invention on IL-12 is more than 20% higher than that of the ubsumizumab standard.

Unless otherwise indicated, all starting materials and reagents used in the examples of the present invention are commercially available.

The present invention has been illustrated by the above embodiments, but it should be understood that the above embodiments are for illustrative and descriptive purposes only and are not intended to limit the invention to the scope of the described embodiments. Furthermore, it will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that many variations and modifications may be made in accordance with the teachings of the present invention, which variations and modifications are within the scope of the present invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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Claims (15)

1. A method of making an anti-IL-12 and/or IL-23 monoclonal antibody or antigen-binding fragment thereof comprising expressing said monoclonal antibody or said antigen-binding fragment using a DHFR knockout or GS knockout CHO cell, said monoclonal antibody or said antigen-binding fragment comprising a heavy chain variable region comprising VH-CDR1, VH-CDR2 and VH-CDR3 having the amino acid sequences shown in SEQ ID No. 1, SEQ ID No.2 and SEQ ID No. 3, respectively; the light chain variable region comprises VL-CDR1, VL-CDR2 and VL-CDR3 which have the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

2. The method of claim 1, wherein the anti-IL-12 and/or IL-23 monoclonal antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID No. 7 and the light chain variable region of SEQ ID No. 8.

3. The method of claim 2, wherein the monoclonal antibody is an ubsunno monoclonal antibody.

4. A method according to any one of claims 1-3, the method comprising the steps of:

a) cloning the coding sequence of the monoclonal antibody into an expression vector to obtain a recombinant expression vector, wherein the expression vector preferably contains a selection marker gene, and more preferably the selection marker gene is a DHFR gene or a GS gene;

b) transferring the recombinant expression vector obtained in the step a) into a CHO cell with a DHFR gene knockout or a CHO cell with a GS gene knockout to obtain a CHO cell modified by genetic engineering;

c) culturing the genetically engineered CHO cell obtained in step b), and selecting a monoclonal cell strain expressing the antibody;

d) culturing the monoclonal cell strain expressing the antibody obtained in the step c), harvesting a cell culture solution containing the antibody, and separating and purifying to obtain the monoclonal antibody.

5. The method of any one of claims 1-4, wherein the DHFR gene knockout CHO cell is a CHO-DG44 cell.

6. The method of claims 1-4, the GS gene knockout CHO cell is treated by employing Zinc Finger Nuclease (ZFN) technology or using CRISPR methodKnocking out GS gene to obtain; preferably, the GS gene knockout CHO cell is

-a GS cell.

7. An anti-IL-12 and/or IL-23 monoclonal antibody or antigen-binding fragment thereof prepared by the method of any one of claims 1-6.

8. A pharmaceutical composition comprising the monoclonal antibody produced by the method of any one of claims 1-6, sucrose, L-histidine hydrochloride monohydrate, and polysorbate 80.

9. The pharmaceutical composition of claim 8, comprising the monoclonal antibody 90 + 9mg/mL, sucrose 76 + 7.6mg/mL, L-histidine 0.5 + 0.3mg/mL, L-histidine hydrochloride monohydrate 1 + 0.5mg/mL, and polysorbate 800.04 + 0.004 mg/mL.

10. The pharmaceutical composition of claim 9, comprising the monoclonal antibody 90mg/mL, sucrose 76mg/mL, L-histidine 0.636mg/mL, L-histidine hydrochloride monohydrate 1.237mg/mL, and polysorbate 800.04 mg/mL.

11. A pharmaceutical composition comprising the monoclonal antibody prepared by the method of any one of claims 1-6, L-histidine hydrochloride monohydrate, polysorbate 80, sucrose, EDTA disodium salt dihydrate, and L-methionine.

12. The pharmaceutical composition of claim 11, comprising said monoclonal antibody 5 + 0.5mg/mL, L-histidine 0.77 + 0.077mg/mL, L-histidine hydrochloride monohydrate 1.03 + 0.10mg/mL, polysorbate 800.4 + 0.04mg/mL, sucrose 85 + 8.5mg/mL, EDTA disodium salt dihydrate 0.02 + 0.002mg/mL, and L-methionine 0.4 + 0.04 mg/mL.

13. The pharmaceutical composition of claim 12, comprising 5mg/mL of the monoclonal antibody, 0.77mg/mL of L-histidine, 1.03mg/mL of L-histidine hydrochloride monohydrate, 800.4 mg/mL of polysorbate, 85mg/mL of sucrose, 0.02mg/mL of EDTA disodium salt dihydrate, and 0.4mg/mL of L-methionine.

14. Use of an anti-IL-12 and/or IL-23 monoclonal antibody or antigen-binding fragment prepared by the method of any one of claims 1-6, or the pharmaceutical composition of any one of claims 8-13, in the manufacture of a medicament for the treatment of an IL-12 and/or IL-23 associated disease or disorder.

15. The use according to claim 14, the disease or disorder is selected from plaque psoriasis, psoriatic arthritis, crohn's disease, systemic lupus erythematosus, ulcerative colitis, multiple sclerosis, sarcoidosis, central spine arthritis, and ankylosing spondylitis; preferably, the disease or disorder is selected from plaque psoriasis, psoriatic arthritis and crohn's disease.