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Anti-interleukin 17 (IL-17A) antibody and application thereof

An antibody and antigen technology, applied in the direction of antibodies, applications, antibody mimics/scaffolds, etc., can solve problems such as weak cell stability, complex expression regulation, tissue damage, etc.

Inactive Publication Date: 2020-06-19
SHANGHAI JUNSHI BIOSCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression and regulation of IL-17A is very complex. Studies have found that cytokines IL-6, IL-1β and TGFβ induce initial CD4 + T cells differentiate into Th17, but at this time Th17 cells have weak stability and secrete a small amount of IL-17A, which has little tissue damage effect; when IL-23 exists, it promotes the stability of Th17 cells and continuously secretes IL-17A, up-regulates and promotes IL-17A. Inflammatory factor (IL-22, CSF-2 and IFN-γ) and down-regulation of anti-inflammatory factor (IL-2, IL-27 and IL-12) expression and other pathways, causing inflammatory outbreak and tissue damage (McGeachy MJ, et al .Natureimmunology.2009,10:314–324)

Method used

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  • Anti-interleukin 17 (IL-17A) antibody and application thereof
  • Anti-interleukin 17 (IL-17A) antibody and application thereof
  • Anti-interleukin 17 (IL-17A) antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0153] Example 1. Recombinant protein human IL-17A-mFc

[0154] The plasmid HG12047-G encoding the full-length human IL-17A cDNA sequence (NCBI accession number: NP_002181.1) was purchased from Sino Biological, and the mature human IL-17A fragment (NCBI accession number NP_002181.1) was amplified by conventional PCR technology. The 24th-155th amino acid, the amino acid sequence is SEQ ID NO.:66, and the nucleotide sequence is SEQ ID NO.:67). After digestion with BSPQI, the amplified fragment was cloned into a self-constructed eukaryotic expression plasmid system (MXT1-Fc, containing the Fc domain of the murine IgG heavy chain) to generate the recombinant fusion protein expression plasmid IL-17A-mFc. Using conventional techniques, the correctly identified plasmid was transfected into expression cell 293F, expressed and purified to obtain human IL-17A-mFc recombinant protein. Such as figure 1 Shown is the SDS-PAGE electrophoresis of human IL-17A-mFc recombinant protein.

Embodiment 2

[0155] Example 2. Construction of 293F stably transfected cell lines expressing human IL-17RA

[0156] The plasmid HG10895-G containing the cDNA sequence encoding the full-length human IL-17RA was purchased from Sino Biological, and the DNA sequence encoding the full-length human IL-17RA (SEQ ID NO.: 69) was amplified by conventional PCR. Using conventional cloning techniques, the amplified fragments were cloned into a self-constructed eukaryotic expression plasmid system (HXP), which contains a puromycin screening system. The successfully constructed IL-17RA recombinant expression plasmid was transfected into 293F (ATCC) cells. After 24 hours of transfection, they were screened by puromycin (2 μg / ml) until the 293F IL-17RA stably transfected cell bank was formed. Use conventional methods to isolate single clones, such as by limiting dilution method, 0.8 cells per well, and spread 96-well plates. After 15 days, select IL-17RA-293F single clones and pass them to form 293F IL-1...

Embodiment 3

[0157] Example 3. Binding of recombinant protein IL-17A-mFc to 293F IL-17RA stably transfected cell line

[0158] FACS experiment was used to detect the binding specificity of recombinant protein IL-17A-mFc to IL-17RA on 293F cells. In brief, cells (293F IL-17RA stably transfected cell line) were prepared into 1×10 6 / ml of cell suspension, add 20ul per well into a 96-well plate, the actual number of cells per well is 2×10 4 For each, the recombinant protein IL-17A-mFc (3ug / ml, 20ul / well; experimental group) or 1% BSA (20ul / well; negative control group) was mixed with the cell suspension, and after incubation at 37 degrees Celsius for 30 minutes, the FACS buffer After elution for 3 times, Anti-mouse IgG (1:200) was added and incubated at room temperature for 30 minutes. After elution with FACS buffer for 3 times, it was detected by flow cytometry, and the mean fluorescence intensity (MFI) of each group was compared. Such as figure 2 As shown, the recombinant protein IL-17...

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PUM

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Abstract

The invention provides an antibody specifically bound to interleukin 17 (IL-17A) with high affinity or a functional fragment of the antibody, and further provides a nucleic acid molecule coding the antibody or the functional fragment thereof, an expression vector used for expressing the antibody or the functional fragment thereof and a host cell, and a production method for the antibody or the functional fragment thereof. The invention further provides a pharmaceutical composition comprising the antibody or the functional fragment thereof, and a method for treating immune dysfunctional diseases by using the antibody or the functional fragment thereof.

Description

technical field [0001] The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind IL-17A. The present invention more particularly relates to antibodies and antigen-binding fragments thereof that inhibit IL-17A-mediated biological activity, compositions and methods for treating related diseases using the antibodies or antigen-binding fragments thereof. The invention relates more particularly to the use of anti-IL-17A antibodies or antigen-binding fragments thereof for the treatment of immunopathological diseases, including autoimmune and inflammatory diseases such as rheumatoid arthritis, psoriasis, Spondylitis, multiple sclerosis, systemic lupus erythematosus (SLE), lupus nephritis or chronic obstructive pulmonary disease, asthma, infectious granuloma, cystic fibrosis or cancer. Background technique [0002] Interleukin 17 (IL-17), also known as CTLA-8 or IL-17A, plays a key coordinating role in the immune system. There are six...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24C12N15/13A61K39/395A61P19/02A61P29/00A61P17/06A61P19/08A61P11/00A61P25/00A61P13/12A61P11/06A61P35/00A61P37/02
CPCC07K16/244A61P19/02A61P29/00A61P17/06A61P19/08A61P11/00A61P25/00A61P13/12A61P11/06A61P35/00A61P37/02A61K2039/505C07K2317/76C07K2317/56C07K2317/565C07K2317/24A61K39/395C07K16/24C07K2317/92C07K2319/30C12N15/63
Inventor 姚剑蒙丹冯辉姚盛武海
Owner SHANGHAI JUNSHI BIOSCI
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