Monoclonal antibody targeting interleukin-23 p19 subunit and application thereof

Abstract

The invention discloses a monoclonal antibody of a targeted interleukin-23p19 subunit and an application of the monoclonal antibody of the targeted interleukin-23p19 subunit. The monoclonal antibody targeting the interleukin 23p19 subunit is obtained through screening and comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises complementary determining regions CDR-H1, CDR-H2 and CDR-H3, and amino acid sequences of the heavy chain variable region are shown as SEQ ID NO: 1-3; the light chain variable region comprises complementary determining regions CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences of the light chain variable region are respectively shown as SEQ ID NO: 4-6. The monoclonal antibody can be selectively combined with a p19 subunit of IL-23 instead of a p40 subunit, and can block the combination of IL-23 and an IL-23 receptor. Therefore, the monoclonal antibody can be used for preparing an IL-23 inhibitor or a medicine for treating related diseases.

Description

Technical Field

[0001] The present invention relates to the development of neutralizing antibodies targeting IL-23 subunits, and in particular to a monoclonal antibody targeting interleukin-23p19 subunit and an application thereof. Background Art

[0002] Interleukin-23 (IL-23) is a heterodimeric cytokine that belongs to the interleukin 12 (IL-12) family of cytokines. IL-23 consists of two disulfide-linked subunits, one is its unique p19 subunit, and the other is the p40 subunit shared with IL-12. The p19 subunit has homology with the p35 subunit of IL-12 and other single-chain cytokines such as IL-6 and IL-11. The p40 subunit is homologous to the extracellular domain of hematopoietic cytokine receptors. Although activated macrophages, dendritic cells, T cells, and endothelial cells all express p19, only activated macrophages and dendritic cells simultaneously express p40, thereby producing IL-23. IL-23 has similar but different biological activities from IL-12. Both IL-12 and IL-23 can induce human T cells to proliferate and produce IFN-γ. IL-12 has effects on both naive and memory T cells in humans, while the effects of IL-23 are limited to memory T cells. IL-12 plays a key role in the development of protective innate and adaptive immune responses and tumor surveillance, and is considered a cytokine that promotes Th1 immune responses, while IL-23 is mainly involved in the regulation of Th17 immune responses. The main function of IL-23 is to stimulate the proliferation and differentiation of Th17 cells by binding to its receptor IL-23R, and to release a variety of proinflammatory factors such as IL-17, IL-21, and IL-22, thereby inducing an inflammatory response. In some autoimmune diseases and chronic inflammatory diseases, such as psoriasis and rheumatoid arthritis, the abnormal expression and activity of IL-23 are highly correlated. Therefore, neutralizing antibodies to IL-23 will inhibit the secretion and proinflammatory effects of IL-17, and ultimately inhibit the effects of IL-17 on inflammatory diseases.

[0003] Therefore, there is a need for a high affinity neutralizing antibody that can specifically bind to the p19 subunit of IL-23 and block binding to IL-23R, thereby blocking the proinflammatory effects of IL-17, and an IL-23 antibody that does not bind to the p40 subunit shared with the related cytokine family member IL-12, which can provide effective treatment for immune-mediated diseases while having limited effects on important host defense immune mechanisms, and such an antibody can be used as a therapeutic agent. Summary of the invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a monoclonal antibody targeting the interleukin-23 p19 subunit.

[0005] Another object of the present invention is to provide a DNA molecule.

[0006] Another object of the present invention is to provide the application of the monoclonal antibody.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A monoclonal antibody targeting the p19 subunit of interleukin 23 (IL-23), comprising a heavy chain variable region and a light chain variable region;

[0009] The heavy chain variable region comprises complementary determining regions CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences thereof are shown in SEQ ID NOs: 1 to 3, respectively;

[0010] The light chain variable region comprises complementary determining regions CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences thereof are shown in SEQ ID NOs: 4 to 6, respectively.

[0011] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7.

[0012] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0013] A DNA molecule (isolated polynucleotide) encoding the above-mentioned monoclonal antibody.

[0014] The monoclonal antibody is used in the preparation of an IL-23 inhibitor and / or an IL-23 molecule detection reagent (or kit).

[0015] An IL-23 inhibitor comprises the above monoclonal antibody.

[0016] An IL-23 molecular detection reagent or kit comprises the above-mentioned monoclonal antibody.

[0017] The monoclonal antibody is used in the preparation of medicines for treating autoimmune diseases and / or inflammatory diseases.

[0018] The autoimmune disease includes at least one of multiple sclerosis, rheumatoid arthritis, psoriasis, graft-versus-host disease, lupus (lupus erythematosus) and metabolic syndrome.

[0019] The inflammatory disease includes chronic inflammatory disease; preferably at least one of inflammatory bowel disease and ankylosing spondylitis.

[0020] The monoclonal antibody is used in preparing medicine for treating cancer.

[0021] The cancer includes at least one of melanoma, colon cancer, ovarian cancer, head and neck cancer, lung cancer, breast cancer or gastric cancer.

[0022] The drug may include, in addition to the above-mentioned monoclonal antibody, one or more pharmaceutically acceptable carriers, diluents or excipients.

[0023] The monoclonal antibody (23A8) of the present invention can bind to the p19 subunit of human IL-23 with a wide range of affinities (KD).

[0024] In some embodiments, the antibodies of the invention may optionally bind to human IL-23 p19 with high affinity. For example, human or other mAbs (monoclonal antibodies) may bind to human IL-23 p19 with an equilibrium dissociation constant (KD) equal to or less than about 10 - 7 M, for example, but not limited to 0.1-9.9 (or any range or value therein)×10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 Or any range or any value therein, can be determined by surface plasmon resonance or Kinexa method.

[0025] In some preferred embodiments, the antibody (23A8) of the present invention binds to human IL-23; or more specifically, the antibody binds to the p19 subunit of IL-23 with a KD of approximately 7.85×10 -7 M to 2.27×10 -9 M.

[0026] Compared with the prior art, the present invention has the following advantages and effects:

[0027] 1. The present invention screened out a neutralizing antibody with IL-23p19 binding specificity and potential therapeutic potential. The IL-23 p19 subunit is a component of the cytokine IL-23, and IL-23 plays an important role in the pathogenesis of various autoimmune diseases, chronic inflammatory diseases, and cancers. Therefore, it can be used to develop related products for the treatment of these related diseases.

[0028] 2. The antibody molecule 23A8 of the present invention is selective for the p19 subunit of human IL-23, and the antibody does not detectably bind to the p40 subunit shared by human IL-23 and human IL-12; when bound to the p19 subunit of human IL-23, the antibody prevents the binding of human IL-23 to the IL-23 subunit of the IL-23 receptor. Therefore, the antibody of the present invention inhibits the activity of human IL-23 on the human IL-23 subunit of the IL-23 receptor.

[0029] 3. The antibody molecule 23A8 in the present invention has similar affinity to the IL-23 p19 antibody molecule Mirikizumab in the prior art and can block the binding of IL-23 to IL-23R. The EC of 23A8 is 50 0.125nM, IC 50 The blocking ability is 0.056 nM, which is similar to the blocking ability of the IL-23 antibody molecule Mirikizumab in the prior art, and has a good application prospect in the preparation of IL-23 inhibitors and the treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a chromatogram of the IL-23 p19 antibody 23A8 of the present invention detected by the HPLC-SEC method.

[0031] Figure 2 It is a binding curve diagram of the antibody 23A8 of the present invention and the antigen IL-23.

[0032] Figure 3 This is a binding curve diagram of the antibody 23A8 of the present invention blocking normal IL-23 / IL-23R.

[0033] Figure 4 The graph shows the experimental results of the specific binding of the antibody 23A8 of the present invention to the rhIL-23p19 monomer (but not the rhIL-23p40 monomer).

[0034] Figure 5 It is a kinetic binding curve diagram of the antibody 23A8 of the present invention and IL-23. DETAILED DESCRIPTION

[0035] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0036] Example 1: Production and screening of mouse antibodies

[0037] Five female BALB / c mice aged 6-8 weeks and weighing 18-25 g (commonly available) were immunized subcutaneously and at multiple points on the footpad with rhIL23A protein (Recombinant Human IL-23P19 / IL23AProtein (mFcTag), Cat: #13062-H05H, Sino Biological) with an Fc tag. 50 μg of antigen was used each time and mixed with Freund's adjuvant in an equal volume of 1:1. Each immunization was performed 7 times with an interval of 1 week to obtain mice with high serum titer. 8×10 5 The spleen cells of the titer mice were fused with SP20 myeloma cells to form hybridoma cells, and the fusion ratio of spleen cells to SP20 myeloma cells was 5:1. Then the binding screening was carried out, and the screening method was "direct ELISA", that is, rhIL23 (Recombinant HumanIL-23 (C-6His), Cat: #CJ40, Novoprotein) with His tag was coated on the ELISA plate, incubated with the culture medium supernatant, and then HRP-labeled sheep anti-mouse IgG antibody (Cat: #115-035-003, Jackson) and TMB substrate were used for colorimetric screening. Through binding screening, a large number of binding clones were obtained. Finally, the antibody with the strongest affinity was selected and named: 23A8.

[0038] Example 2: Sequence analysis of mouse antibodies

[0039] The cDNA of the light chain and heavy chain of 23A8 was cloned and sequenced. The sequence was analyzed using the tools on AbYsis combined with manual experience, and the framework region, complementary determining region (CDR) and invariant region were annotated. The variable region of 23A8 (through the framework region and CDR region) was searched for homology with the Genebank protein sequence, and the results showed that the framework region was highly homologous to the framework region of various mouse antibodies. No protein sequence homologous to the CDR region of 23A8 was found. Therefore, 23A8 is a completely new mouse antibody whose sequence has not been reported yet.

[0040] The sequencing results of the 23A8 light chain and heavy chain variable regions are as follows:

[0041] CDR-H1: SYGVN (SEQ ID NO: 1);

[0042] CDR-H2: AIWAGGSTNYNSALMS (SEQ ID NO: 2);

[0043] CDR-H3: EAMDY (SEQ ID NO:3);

[0044] CDR-L1: KASQDVGTAVA (SEQ ID NO: 4);

[0045] CDR-L2: WASVRHF (SEQ ID NO:5);

[0046] CDR-L3: QQYSSYPYT(SEQ ID NO:6);

[0047] 23A8 heavy chain variable region (SEQ ID NO: 7):

[0048] EVKLKESGPGLVAPSQSLSITCTVSGFSLTSYGVNWVRQPPGKGLEWLGAIWAGGSTN YNSALMSRLSISKDNSKSQVLLKMNSLQTDDIAMYYCAREAMDYWGQGTSVIVSS;

[0049] 23A8 light chain variable region (SEQ ID NO: 8):

[0050] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKARQSPNLLIYWASVRHFG VPDRFTGSGSGTDFTLTVSNVQSEDLADYFCQQYSSYPYTFGGGTKLEIK.

[0051] Example 3: HPLC-SEC detection of the purity of IL-23p19 antibody 23A8

[0052] (1) Antibody preparation: The monoclonal antibody in Example 1 was expanded to 50 mL and cultured with shaking for 5 days until the cell survival rate dropped to about 30%. Collect the supernatant by centrifugation at 4000 rpm*10 min in a high-speed centrifuge. After incubation with protein A filler (rProtein ABeads, Cat:#SA012025, Tiandirenhe Biotechnology Co., Ltd.) for 1 hour, the supernatant was eluted with 100 mM glycine at pH 1.5 to obtain the purified antibody. Dilute the antibody to 1 mg / ml with PBS, take 30 μl for later use, centrifuge at 12000g for 2 minutes, try to remove any possible precipitate, then aspirate the supernatant after centrifugation and add it to the inner tube, remove all bubbles, and wait for loading.

[0053] (2) Preparation of mobile phase: Prepare 50 mM phosphate buffer for HPLC, adjust the pH to 7.0, and sterilize the buffer by vacuum filtration. Then, place the buffer in an ultrasonicator for ultrasonic degassing and set aside.

[0054] (3) Instrument preparation: Turn on the instrument, then open each module of the HPLC, and perform preheating. First, remove bubbles from the instrument system, then connect an empty column and clean the entire instrument with sterile water. After cleaning, connect the Agilent SEC column to the instrument, remove bubbles, and then clean the column with sterile water. After cleaning, balance the column with equilibration buffer and set aside.

[0055] (4) Sampling: Open the data acquisition software, input the sample information, set the sample flow rate to 1 ml / min, and use a 280 nm UV detector to obtain the signal value, and then perform sample loading.

[0056] (5) Result analysis and processing: Open the software, import the results, perform analysis, and generate an analysis report.

[0057] Figure 1 The results showed that the purity of the antibody 23A8 prepared by the present invention was about 100% according to the chromatogram detected by HPLC-SEC.

[0058] Example 4: Antigen Binding Analysis

[0059] (1) Coating: Dilute the His-tagged human cytokine interleukin-23 protein (Recombinant Human IL-23 (C-6His), Cat: #CJ40, Novoprotein) to 2 μg / mL in PBS (pH 7.2), coat a 96-well high-adsorption U-shaped plate (Greiner Bio-One North America Inc., Monroe, NC, USA) at 50 μL / well, and incubate at 4°C overnight.

[0060] (2) Washing: Wash three times with PBS.

[0061] (3) Blocking: The 96-well plate was blocked with blocking solution (1% casein, Thermo Fisher Scientific, Waltham, MA, USA) at 200 μL / well and left at room temperature for 1.5 hours.

[0062] (4) Washing: Wash three times with 0.1% PBST.

[0063] (5) Primary antibody: 23A8 antibody purified in Example 3 diluted with blocking solution at a gradient dilution ratio of 1:3 was added (exemplary concentrations are: 20 μg / ml, 6.6 μg / ml, 3.3 μg / ml, 0.74 μg / ml, 0.24 μg / ml, 0.082 μg / ml, 0.028 μg / ml, 0.009 μg / ml, 0.003 μg / ml, 0.001 μg / ml), and the sample included a positive control antibody targeting IL-23p19 (prepared by the method disclosed in Example 1 of WO2014137962A1, which was developed by Eli Lilly and Company of the United States and has a trade name of Mirikizumab) (named PC), and incubated at room temperature for 1 hour at 50 μL / well. At the same time, a blank control (NC) was used without adding any antibody.

[0064] (6) Washing: Wash three times with 0.1% PBST.

[0065] (7) Secondary antibody: HRP Goat AntiMouse IgG (H+L) (ABclonal Technology Co., Ltd., Wuhan, China) diluted in blocking solution (1% casein) at a ratio of 1:2000 (v / v) was added and incubated at room temperature for 1 hour at 50 μL / well.

[0066] (8) Washing: Wash three times with 0.1% PBST.

[0067] (9) Color development: Add 50 μL / well of TMB solution (Thermo Fisher Scientific) and incubate at room temperature for 5 min.

[0068] (10) Termination: Add 50 μL / well of 2 M sulfuric acid to terminate the reaction.

[0069] (11) Data analysis: The absorbance at a wavelength of 450 nm was read using a microplate reader SpectraMax M5e (Molecular Devices, LLC, San Jose, CA, USA), and data were analyzed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA) to generate the corresponding binding curve. The experiment was repeated three times.

[0070] Figure 2 The results showed that the affinity of 23A8 to the antigen was 0.125 nM.

[0071] Example 5: 23A8 antibody molecule blocks the binding of IL-23 to IL-23R

[0072] (1) Coating: Dilute the cytokine interleukin 23 receptor protein (rhIL-23R) (Recombinant Human IL-23 Receptor Protein (ECD, hFc Tag), Cat: #13840-H02H, Sino Biological) to 1 μg / mL with PBS (pH 7.2), coat a 96-well high-adsorption U-shaped plate at 50 μL / well, and incubate at 4°C overnight.

[0073] (2) Washing: Wash three times with PBS.

[0074] (3) Blocking: Block the 96-well plate with blocking solution (1% casein) at 200 μL / well and leave at room temperature for 2 hours.

[0075] (4) Washing: Wash three times with 0.1% PBST.

[0076] (5) Antigen-antibody preincubation: Antibody 23A8 (the purified antibody in Example 3) and positive antibody PC (Mirikizumab, same as in Example 4) (exemplary concentrations: 10 μg / ml, 6.7 μg / ml, 4.4 μg / ml, 3.0 μg / ml, 2.0 μg / ml, 1.3 μg / ml, 0.88 μg / ml, 0.59 μg / ml, 0.39 μg / ml, 0.26 μg / ml, 0.17 μg / ml) diluted in blocking solution at a gradient dilution ratio of 1:1.5 (v / v) were mixed with 1 μg / ml biotinylated rhIL23-His (Recombinant Human IL-23 (C-6His), Cat: #CJ40, Novoprotein) in equal volumes and incubated at room temperature for 2 h.

[0077] (6) Antigen-antibody incubation: The incubated antigen-antibody mixture was added into the U-shaped plate at 50 μl / well and incubated at room temperature for 1 h.

[0078] (7) Washing: Wash three times with 0.1% PBST.

[0079] (8) Secondary antibody: HRP-labeled streptavidin (Thermo Fisher Scientific) diluted in blocking solution (1% casein) at a ratio of 1:2500 (v / v) was added and incubated at room temperature for 1 hour at 50 μL / well.

[0080] (9) Washing: Wash three times with 0.1% PBST.

[0081] (10) Color development: Add 50 μL / well of TMB solution (Thermo Fisher Scientific) and incubate at room temperature for 5 minutes

[0082] (11) Termination: Add 50 μL / well of 2 M sulfuric acid to terminate the reaction.

[0083] (12) Data analysis: The absorbance value at a wavelength of 450 nm was read using a SpectraMax M5e microplate reader, and data analysis was performed using GraphPad Prism 9 to generate the corresponding IL-23 and IL-23R binding blocking curve. The experiment was repeated three times.

[0084] Figure 3 The results showed that 23A8 blocked the IC of IL-23 binding to IL-23R 50 The blocking ability was 0.056 nM, which was similar to that of the positive control antibody PC (Mirikizumab, Eli Lilly).

[0085] Example 6: 23A8 molecule specifically binds to rhIL23 p19 subunit but not to rhIL23 p40 subunit

[0086] (1) Coating: rhIL-23A&B (Novoprotein), rhIL23A-Fc (i.e., IL23p19 subunit, Sino Biological), and rhIL23B (i.e., IL23p40 subunit, Sino Biological) proteins with His tags were diluted to 2 μg / mL in PBS at pH 7.2, and 50 μL / well was used to coat a 96-well high-adsorption U-shaped plate and incubated at 4°C overnight. The blank control (NC) was used without adding any antibody.

[0087] (2) Washing: Wash three times with PBS.

[0088] (3) Blocking: Block the 96-well plate with blocking solution (1% casein) at 200 μL / well and leave at room temperature for 1.5 hours.

[0089] (4) Washing: Wash three times with 0.1% PBST.

[0090] (5) Primary antibody: 1 μg / ml of 23A8 antibody (the purified antibody in Example 3) and positive control antibody PC (Mirikizumab, same as in Example 4) were added respectively, and incubated at room temperature for 1 hour at 50 μL / well.

[0091] (6) Washing: Wash three times with 0.1% PBST.

[0092] (7) Secondary antibody: HRP Goat AntiMouse IgG (H+L) (ABclonal Technology Co., Ltd., Wuhan, China) diluted in blocking solution (1% casein) at a ratio of 1:2000 (v / v) was added and incubated at room temperature for 1 hour at 50 μL / well.

[0093] (8) Washing: Wash three times with 0.1% PBST.

[0094] (9) Color development: Add 50 μL / well of TMB solution (Thermo Fisher Scientific) and incubate at room temperature for 5 min.

[0095] (10) Termination: Add 50 μL / well of 2 M sulfuric acid to terminate the reaction.

[0096] (11) Data analysis: The absorbance at a wavelength of 450 nm was read using a SpectraMax M5e microplate reader, and data were analyzed using GraphPadPrism 9. The experiment was repeated three times.

[0097] Figure 4 The results showed that the 23A8 antibody specifically bound to the p19 subunit of rhIL-23 but not to the p40 subunit, which was similar to the antigen binding characteristics of the positive control antibody PC (Mirikizumab, Eli Lilly).

[0098] Example 7: Affinity analysis of 23A8 and IL-23

[0099] Using FortéBio The affinity analysis of each fusion protein and rhIL23A&B-His (Sino Biological) was performed using a Pall FortéBio LLC, Fremont, CA, USA instrument. The 23A8 antibody (10 μg / mL) was captured by a Protein G probe (Pall FortéBio LLC), and the interaction activity of the 23A8 antibody at different concentrations of 200 nM, 100 nM, and 50 nM was detected in 0.1% PBST universal buffer. The affinity constants of 23A8 and IL23A&B-His protein were calculated based on the affinity and kinetics 1:1 binding mode fitting in Octet Analysis software. The experiment was repeated three times.

[0100] Figure 5 The results showed that the KD value of 23A8 and IL23A&B was 7.85×10 -7 M.

[0101] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A monoclonal antibody targeting interleukin 23p19 subunit, characterized in that: comprising a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises complementary determining regions CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences thereof are shown in SEQ ID NOs: 1 to 3, respectively; The light chain variable region comprises complementary determining regions CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences thereof are shown in SEQ ID NOs: 4 to 6, respectively.

2. The monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. A DNA molecule, characterized in that: Encodes the monoclonal antibody according to claim 1 or 2.

4. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of an IL-23 inhibitor and / or an IL-23 molecule detection reagent or kit.

5. An IL-23 inhibitor, characterized in that: Comprising the monoclonal antibody according to claim 1 or 2.

6. An IL-23 molecular detection reagent or kit, characterized in that: Comprising the monoclonal antibody according to claim 1 or 2.

7. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a medicament for treating autoimmune diseases and / or inflammatory diseases.

8. The use according to claim 7, characterized in that: The autoimmune disease includes at least one of multiple sclerosis, rheumatoid arthritis, psoriasis, graft-versus-host disease, lupus and metabolic syndrome; The inflammatory diseases include chronic inflammatory diseases.

9. The use according to claim 8, characterized in that: The inflammatory disease is at least one of inflammatory bowel disease and ankylosing spondylitis.

10. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a drug for treating cancer.

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