Anti-IL-23R antibody and application thereof

An antibody and antigen technology, applied in the direction of antibodies, antibody medical components, applications, etc., can solve problems such as unsatisfactory properties

Active Publication Date: 2021-11-26
HISUN BIORAY PHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the properties of 20D7 molecules are not up to expectations, follow-up clinical research has not been carried out for many years. Therefore, there is an urgent need to develop antibodies with better properties in this field.

Method used

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  • Anti-IL-23R antibody and application thereof
  • Anti-IL-23R antibody and application thereof
  • Anti-IL-23R antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1 Preparation of anti-IL-23 antibody mouse monoclonal antibody

[0064] 6-8 week old BALB / c mice (commercially purchased) were first immunized with 50 μg of commercial human IL-23R-ECD-Fc protein (Sino Biological, Cat. No. H02H). On the 14th day and 28th day after the first immunization, the immunized mice were immunized again with 25 μg IL-23R-ECD-Fc protein. The serum titer of immunized mice was detected by enzyme-linked immunosorbent assay (Elisa). The IL-23R-His (ACROBiosystem, H52H4) antigen was diluted to 0.5 μg / ml with PBS, and coated on a microwell plate overnight at 4°C. Then 1% BSA-PBS blocking solution was used to block at 37°C for 1h; after washing the plate with PBST, serum dilutions from mice were added to the plate and incubated at 37°C for 1hr. Wash the plate and add HRP-labeled goat anti-mouse IgG (Sigma, A0170) (1:10000), react at 37°C for 0.5h, wash the plate and add TMB solution, react at room temperature for 5 minutes in the dark, add 2...

Embodiment 2

[0066] Example 2 Detection of binding of antibody to human IL-23R

[0067] The binding ability of the purified antibody to IL-23R protein was detected by Elisa method. The specific method was as described in Example 1, wherein the initial concentration of the mouse antibody was 740 pM, and a total of 10 gradients were obtained by 3-fold serial dilution with PBS. Each antibody was tested in duplicate wells, and the EC50 was analyzed by curve fitting. As a result, it was confirmed that the antibodies named mAb#5D4, mAb#5E2, mAb#7A12, mAb#2C5, and mAb#9A10 had high binding activity to human IL-23R ( figure 1 ).

[0068] Flow cytometry (FCM) was used to analyze the binding of the above mouse monoclonal antibodies to IL-23R on the membrane of 293 cells 293-IL23 Res (Novoprotein, XCC05) that exogenously overexpressed IL-23R. The cells were reacted with different concentrations of monoclonal antibodies (the highest concentration was 11.11 nM, 3-fold serial dilution, a total of 9 co...

Embodiment 3

[0069] Example 3 Blocking Effect of Antibodies on the Binding of IL-23R and IL-23 Ligand

[0070] It was further tested whether these antibodies could block the binding of IL-23R and IL-23. IL-23R (ACROBiosystem, ILR-H5254) was diluted to 1 μg / mL, added to the microtiter plate at 100 μL / well, and coated overnight at 4°C. After washing the plate, 240 μL of 5% BSA-PBST was added to each well and blocked at 37°C for 1 hour. Dilute biotin-labeled IL-23α & IL-12β heterodimer protein (ACROBiosystem, ILB-H82W6) to 0.2μg / mL with diluent (1×PBST); dilute each antibody sample to 66.7nM, and then perform a 3-fold gradient Diluted to obtain a total of 10 concentration points. Add 50 μL of serially diluted antibody samples and 50 μL of IL-23α & IL-12β to each well, place at 37°C for 1 hour and wash the plate three times, add 100 μL of HRP-streptavidin (Abcam, Ab7403) to each well after dilution, and horizontally place at 37°C After standing for 1 hour, wash 3 times. Add 100 μL TMB chro...

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Abstract

The invention relates to a novel antibody of an interleukin-23 receptor or an antibody fragment thereof. The antibody or the fragment thereof can be effectively combined with IL-23R and block combination of IL-23R and a human IL-23 alpha / IL-12 beta heterodimer ligand, and has a good application prospect. The antibody or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3; the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3; the VH CDR1 comprises an amino acid sequence shown in SEQ ID NO: 3, 13 or 23; the VH CDR2 comprises an amino acid sequence shown in SEQ ID NO: 4, 14 or 24; the VH CDR3 comprises an amino acid sequence shown in SEQ ID NO: 5, 15 or 25; the VL CDR1 comprises an amino acid sequence shown in SEQ ID NO: 8, 18 or 28; the VL CDR2 comprises an amino acid sequence shown in SEQ ID NO: 9, 19 or 29; and the VL CDR3 comprises an amino acid sequence shown in SEQ ID NO: 10, 20 or 30.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to antibodies and applications thereof. Background technique [0002] Interleukin 23 (IL-23) is composed of two protein subunits, namely IL-23-specific p19 subunit (IL-23α) and p40 subunit (IL-12β, p40 subunit is used together with IL-12) Heterodimeric cytokines are generally produced by activated macrophages or dendritic cells and act on Th17 cells expressing IL-23 receptors to promote their proliferation and stability. [0003] IL-23 induced CD4+ T cells to produce IL-17 was mediated by activated Jak2, PI3K / Akt, STAT3 and NF-κB. Tyk2 and other members of the STAT family such as STAT1, STAT4, STAT5 are also involved in this process. The IL-23 receptor is composed of IL-12Rβ1 subunit and IL-23R subunit, forming a heterodimer, in which IL-12Rβ1 binds to Tyk2, and IL-23R binds to Jak2. IL-23 binds to its receptor complex and activates its downstream Jak2 and Tyk2, leading to phosphorylation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61K45/06A61P37/02A61P1/00A61P29/00A61P1/04A61P17/06A61P19/02A61P19/08
CPCC07K16/2866A61K39/3955A61K45/06A61P37/02A61P1/00A61P29/00A61P1/04A61P17/06A61P19/02A61P19/08C07K2317/565C07K2317/56C07K2317/51C07K2317/515C07K2317/76A61K2039/505A61K2300/00
Inventor 王晓泽吴振华徐统聂磊梅小芬陈刚王海彬陈旭晨潘晨晓
Owner HISUN BIORAY PHARMA CO LTD
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