Monoclonal antibodies targeting interleukin-23 p19 subunit and uses thereof

Abstract

The application discloses a monoclonal antibody targeting interleukin-23 p19 subunit and application thereof. The application obtains a monoclonal antibody targeting interleukin-23 p19 subunit through screening, and the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementary determining regions CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences 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 are shown as SEQ ID NO:4-6. The monoclonal antibody can selectively combine with the p19 subunit of IL-23 without combining with the p40 subunit, and can block the combination of IL-23 and IL-23 receptor. Therefore, the monoclonal antibody can be used for preparing an IL-23 inhibitor or a drug for treating related diseases.

Description

Technical Field

[0001] This invention relates to the development of neutralizing antibodies against the IL-23 subunit, and particularly to a monoclonal antibody targeting the interleukin-23p19 subunit and its application. Background Technology

[0002] Interleukin-23 (IL-23) is a heterodimeric cytokine belonging to the interleukin-12 (IL-12) family of cytokines. IL-23 consists of two disulfide-linked subunits: its unique p19 subunit and the p40 subunit shared with IL-12. The p19 subunit is homologous to the p35 subunit of IL-12, as well as 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, thus producing IL-23. IL-23 possesses biological activities similar to but different from IL-12. Both IL-12 and IL-23 can induce human T cell proliferation and IFN-γ production. IL-12 affects both naive T cells and memory T cells in humans, while IL-23's effects are limited to memory T cells. IL-12 plays a crucial role in the development of protective innate and adaptive immune responses and in tumor surveillance, and is considered a cytokine that promotes Th1 immune responses, while IL-23 primarily participates 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, releasing various pro-inflammatory factors such as IL-17, IL-21, and IL-22, thereby triggering an inflammatory response. Abnormal expression and activity of IL-23 are highly correlated in some autoimmune diseases and chronic inflammatory diseases, such as psoriasis and rheumatoid arthritis. Therefore, neutralizing antibodies against IL-23 will inhibit the secretion and pro-inflammatory effects of IL-17, ultimately suppressing the influence of IL-17 on inflammatory diseases.

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

[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a monoclonal antibody that targets 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 aforementioned monoclonal antibody.

[0007] The objective of this invention is achieved through the following technical solution:

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

[0009] The heavy chain variable region includes complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, whose amino acid sequences are shown in SEQ ID NO: 1 to 3, respectively;

[0010] The light chain variable region includes complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, whose amino acid sequences are shown in SEQ ID NO: 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 aforementioned monoclonal antibody.

[0014] The application of the monoclonal antibody in the preparation of IL-23 inhibitors and / or IL-23 molecular detection reagents (or kits).

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

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

[0017] The use of the monoclonal antibody in the preparation of medicaments for the treatment of autoimmune diseases and / or inflammatory diseases.

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

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

[0020] The application of the monoclonal antibody in the preparation of drugs for treating cancer.

[0021] The cancers mentioned include at least one of melanoma, colon cancer, ovarian cancer, head and neck cancer, lung cancer, breast cancer, or stomach cancer.

[0022] In addition to the monoclonal antibodies mentioned above, the drugs may also include one or more pharmaceutically acceptable carriers, diluents, or excipients.

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

[0024] In some embodiments, the antibody of the present 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 having 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 any value therein) × 10 -7 10 -8 10 -9 10 -10 10 -11 10 -12 10 -13 Or any range or value thereof, which can be determined by surface plasmon resonance or the 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, which has a KD of approximately 7.85 × 10⁻⁶. -7 M to 2.27×10 -9 M.

[0026] The present invention has the following advantages and effects compared with the prior art:

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

[0028] 2. The antibody molecule 23A8 in this 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 it binds to the p19 subunit of human IL-23, the antibody prevents human IL-23 from binding to the IL-23 subunit of the IL-23 receptor. Therefore, the antibody of this 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 this invention has a similar affinity to the existing IL-23 p19 antibody molecule Mirikizumab, and can block the binding of IL-23 to IL-23R. The EC50 of 23A8... 50 0.125nM, IC 50 With a concentration of 0.056 nM, it has a similar blocking ability to the existing IL-23 antibody molecule Mirikizumab, and has great potential for application in the preparation of IL-23 inhibitors and in the treatment of related diseases. Attached Figure Description

[0030] Figure 1 The chromatogram of IL-23 p19 antibody 23A8 in this invention is obtained by HPLC-SEC method.

[0031] Figure 2 This is a binding curve of antibody 23A8 and antigen IL-23 in this invention.

[0032] Figure 3 This is a graph showing the binding curve of antibody 23A8 in this invention to block normal IL-23 / IL-23R.

[0033] Figure 4 This is a diagram showing the experimental results of the specific binding of antibody 23A8 in this invention to rhIL-23p19 monomer (but not rhIL-23p40 monomer).

[0034] Figure 5 This is a kinetic binding curve of antibody 23A8 and IL-23 in this invention. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

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

[0037] Five 6-8 week old, 18-25g female BALB / c mice (commonly available) were immunized subcutaneously and at multiple sites on their footpads with Fc-tagged rhIL23A protein (Recombinant Human IL-23P19 / IL23A Protein (mFcTag), Cat:#13062-H05H, Sino Biological). Each immunization used 50 μg of antigen, mixed with Freund's adjuvant in a 1:1 ratio. Immunizations were administered one week apart, for a total of seven immunizations, resulting in mice with high serum titers. 8 × 10⁸ mice were collected. 5 Hybridoma cells were formed by fusing spleen cells from high-titer mice with SP20 myeloma cells at a ratio of 5:1. Binding selection was then performed using a direct ELISA method. This involved coating an ELISA plate with a His-tagged rhIL23 (Recombinant HumanIL-23(C-6His), Cat:#CJ40, Novoprotein), incubating it with the culture supernatant, and then using HRP-labeled goat anti-mouse IgG antibody (Cat:#115-035-003, Jackson) and TMB substrate for colorimetric selection. A large number of binding clones were obtained through binding selection. Finally, the antibody with the strongest affinity was selected and named 23A8.

[0038] Example 2: Sequence analysis of murine antibodies

[0039] The light and heavy chain cDNAs of 23A8 were cloned and sequenced. Sequence analysis was performed using tools on AbYsis combined with manual experience, identifying the frame region, complementarity-determining region (CDR), and invariant region. Genebank sequence homology searches were conducted on the variable regions of 23A8 (by examining the frame and CDR regions), revealing high homology between the frame region and the frame regions of various mouse antibodies. No protein sequences homologous to the CDR region of 23A8 were found. Therefore, 23A8 is a novel mouse antibody sequence that has not been previously reported.

[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 from Example 1 was expanded to 50 mL and cultured with shaking for 5 days until the cell viability decreased to about 30%. The supernatant was collected by centrifugation at 4000 rpm for 10 min. The supernatant was co-incubated with protein A packing material (rProtein ABeads, Cat:#SA012025, Tiandi Renhe Biotechnology Co., Ltd.) for 1 h, and then eluted with 100 mM glycine at pH 1.5 to obtain the purified antibody. The antibody was diluted with PBS to 1 mg / mL, and 30 μl was taken for use. The supernatant was centrifuged at 12000 g for 2 min to remove any possible precipitation. The supernatant was then aspirated and added to the insert tube, and air bubbles were removed before loading.

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

[0054] (3) Instrument preparation: Turn on the instrument, then turn on each module of the high performance liquid chromatography and perform a preheating process. First, remove air 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 air bubbles, and then clean the column with sterile water. After cleaning, equilibrate the column with equilibration buffer and set it aside for use.

[0055] (4) Sample loading: 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 load the sample.

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

[0057] Figure 1 The results showed that the antibody 23A8 prepared in this invention had a purity of approximately 100% as determined by HPLC-SEC.

[0058] Example 4: Antigen Binding Ability Analysis

[0059] (1) Coating: The human cytokine interleukin-23 protein (Recombinant Human IL-23 (C-6His), Cat:#CJ40, Novoprotein) with the His tag was diluted to 2 μg / mL with PBS at pH 7.2 and coated with 50 μL / well of 96-well high-adsorption U-shaped plates (Greiner Bio-One North America Inc., Monroe, NC, USA) and incubated overnight at 4°C.

[0060] (2) Washing: Wash 3 times with PBS.

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

[0062] (4) Washing: Wash 3 times with 0.1% PBST.

[0063] (5) Primary antibody: 23A8 antibody purified in Example 3 was added in a 1:3 gradient with blocking buffer (example concentrations: 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). The samples included a positive control antibody targeting IL-23p19 (prepared by the method in Example 1 disclosed in WO2014137962A1, which was developed by Eli Lilly and Company and marketed as Mirikizumab) (named PC). The antibody was incubated at 50 μL / well at room temperature for 1 hour, with a blank control (NC) without any antibody added.

[0064] (6) Washing: Wash 3 times with 0.1% PBST.

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

[0066] (8) Washing: Wash 3 times with 0.1% PBST.

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

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

[0069] (11) Data analysis: The absorbance values ​​at 450 nm were read using a SpectraMax M5e microplate reader (Molecular Devices, LLC, San Jose, CA, USA). Data analysis was performed using a GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA) to generate the corresponding binding curves. The experiment was conducted in triplicate.

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

[0071] Example 5: 23A8 antibody molecules block the binding of IL-23 to IL-23R.

[0072] (1) Coating: The cytokine interleukin-23 receptor protein (rhIL-23R) (ECD, hFc Tag), Cat:#13840-H02H, Sino Biological) was diluted to 1 μg / mL with PBS at pH 7.2 and coated with 50 μL / well of a 96-well high-adsorption U-shaped plate. The plate was incubated overnight at 4°C.

[0073] (2) Washing: Wash 3 times with PBS.

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

[0075] (4) Washing: Wash 3 times with 0.1% PBST.

[0076] (5) Antigen-antibody pre-incubation: Antibody 23A8 (antibody purified in Example 3) and positive antibody PC (Mirikizumab, same as in Example 4) were serially diluted with blocking buffer at a ratio of 1:1.5 (v / v) and 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) Incubation of antigen and antibody: The incubated antigen and antibody mixture was added to a U-shaped plate at 50 μl / well and incubated at room temperature for 1 h.

[0078] (7) Washing: Wash 3 times with 0.1% PBST.

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

[0080] (9) Washing: Wash 3 times with 0.1% PBST.

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

[0082] (11) Termination: The reaction was terminated by adding 50 μL of 2M sulfuric acid per well.

[0083] (12) Data analysis: The absorbance values ​​at 450 nm were read using a SpectraMax M5e microplate reader, and the data were analyzed using a GraphPad Prism9 to generate the corresponding IL-23 and IL-23R binding blocking curves. The experiment was set up in triplicate.

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

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

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

[0087] (2) Washing: Wash 3 times with PBS.

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

[0089] (4) Washing: Wash 3 times with 0.1% PBST.

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

[0091] (6) Washing: Wash 3 times with 0.1% PBST.

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

[0093] (8) Washing: Wash 3 times with 0.1% PBST.

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

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

[0096] (11) Data analysis: The absorbance values ​​at 450 nm were read using a SpectraMax M5e microplate reader, and the data were analyzed using a GraphPad Prism 9. The experiment was set up in triplicate.

[0097] Figure 4 The results showed that the 23A8 antibody specifically binds to the p19 subunit of rhIL-23 but not the p40 subunit, which is 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 Affinity analysis of various fusion proteins with rhIL23A&B-His (Sino Biological) was performed using an instrument (Pall FortéBio LLC, Fremont, CA, USA). A Protein G probe (Pall FortéBio LLC) captured 23A8 antibody (10 μg / mL), and the interaction activity of 23A8 antibody at different concentrations of 200 nM, 100 nM, and 50 nM was detected in 0.1% PBST universal buffer. The affinity constants between 23A8 and IL23A&B-His protein were calculated based on the 1:1 binding pattern fitting in Octet Analysis software. The experiment was performed in triplicate.

[0100] Figure 5 The results showed that the KD value of 23A8 and IL23A&B was 7.85 × 10⁻⁶. -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 changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody targeting the interleukin-23 p19 subunit, characterized in that: It includes variable regions for heavy chains and variable regions for light chains; The heavy chain variable region includes complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, whose amino acid sequences are shown in SEQ ID NO: 1 to 3, respectively; The light chain variable region includes complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, whose amino acid sequences are shown in SEQ ID NO: 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: Encoding the monoclonal antibody as described in claim 1 or 2.

4. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of IL-23 molecular detection reagents or kits.

5. An IL-23 molecular detection reagent or kit, characterized in that: It contains the monoclonal antibody as described in claim 1 or 2.

6. The 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, characterized in that: The autoimmune disease mentioned is at least one of multiple sclerosis, rheumatoid arthritis, psoriasis, and graft-versus-host disease; The inflammatory disease mentioned is inflammatory bowel disease.

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