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Development and application of therapeutic agent for TSLP-related diseases

A sequence, CDR2 technology, used in applications, fermentation, anti-inflammatory agents, etc.

Active Publication Date: 2021-06-01
CHENGDU CONMED BIOSCI CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In addition, studies have shown that TSLP may promote TH1 / TH17-related autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis

Method used

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  • Development and application of therapeutic agent for TSLP-related diseases
  • Development and application of therapeutic agent for TSLP-related diseases
  • Development and application of therapeutic agent for TSLP-related diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0179] The preparation of embodiment 1, TSLP and TSLPR recombinant protein

[0180]The cDNAs of human TSLP (Uniprot: Q969D9, SEQ ID NO: 1) and cynomolgus monkey TSLP (Uniprot: A0A2K5TXV0, SEQ ID NO: 2) were synthesized, and the sequences were cloned into the eukaryotic expression vector pCMV3 (purchased from Beijing Sinobiological, Cat. No. CG90911 -UT), insert the signal peptide MDMRVPAQLLGLLLLWLRGARS at the N-terminus, and express a tag containing 6 histidines at the carboxy-terminal of TSLP to obtain pSect-hTSLP-cHis and pSect-cyTSLP-cHis plasmids, and transfect HEK293 with these plasmids. 6E cells (ATCC CRL-1573), the cell supernatant was collected, but the recombinant protein of corresponding length was not obtained by nickel column affinity chromatography. Then the full-length fragment of TSLP was cloned into the prokaryotic expression plasmid pET-30a, and the plasmid was transformed into BL21 E.coli. After induction, the recombinant protein was found to be expressed in ...

Embodiment 2

[0183] Embodiment 2, TSLPR stably transfected cell construction

[0184] 1) Construction of Ba / F3-hTSLPR stably transfected cell line

[0185] Ba / F3 cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum, 50 μM 2-mercaptoethanol, 2 mM L-glutamine, 50 μg / mL penicillin-streptomycin and 10 ng / mL mouse IL-3 . The lentiviral expression vector expressing human TSLPR (Guangzhou Funeng Gene Co., Ltd., EX-W0156-Lv105-B) was packaged with the Lenti-Pac HIV lentiviral packaging kit (Guangzhou Funeng Gene Co., Ltd.) and transfected Ba / F3 cells were added with puromycin for resistance selection to obtain a cell line Ba / F3-hTSLPR capable of stably expressing human TSLPR. Figure 4 It was shown that by detecting the binding of biotinylated human TSLP protein to TSLPR on the cell surface, it was confirmed that the constructed cells expressed human TSLPR, and its EC 50 = 0.22 nM.

[0186] 2) Construction of Ba / F3-hTSLPR-IL7Rα stably transfected cell line

[0187] IL-7...

Embodiment 3

[0191] Embodiment 3, animal immunization

[0192] Select 6-week-old female Balb / C mice for immunization. Each mouse is immunized with 50 μg of antigen each time. The antigen is alternately immunized with human and cynomolgus TSLP. The antigen is mixed with Freund's adjuvant in equal volumes and subcutaneously immunized every two weeks. Immune once. After four times of immunization, blood was taken from the tail, and the titer of mouse serum and the inhibitory effect of serum on the combination of TSLP and TSLPR were detected by ELISA. Or after the initial immunization, use a gene gun to boost immunization every other week, mix the human TSLP expression plasmid with gold powder, and bombard the naked skin of the abdomen of the mouse with a pressure of 400 psi, and immunize nine times in total.

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Abstract

The invention discloses development and an application of a therapeutic agent for TSLP-related diseases, and relates to an antibody binding to TSLP protein or an antigen binding part thereof as well as a preparation method and an application of the antibody. The antibody can be combined with human TSLP and / or cynomolgus monkey TSLP with high affinity, can block the combination of the TSLP and TSLPR, and can inhibit the transduction of a TSLP stimulating signal through an STAT5 pathway.

Description

technical field [0001] The present disclosure relates to an antibody recognizing TSLP protein, its production method and application. Background technique [0002] Thymic Stromal Lymphopoietin (TSLP) is a cytokine of the interleukin-7 family, which is mainly produced by epithelial cells and keratinocytes located in the skin, intestinal tract or lungs, and is involved in maintaining the immune system of the mucosa Stablize. After epithelial tissue is stimulated or destroyed by allergens or pathogens, dendritic cells present antigens to naïve CD4+ T cells, and at the same time release epithelial cytokines, TSLP and IL-33. These important costimulatory cytokines lead to the development of allergen-specific TH2 cells, which then produce the cytokines interleukins IL-4, IL-5, and IL-13. This group of TH2-derived cytokines plays a key role in the pathogenesis of allergic diseases. [0003] TSLP is a multifunctional cytokine that can exert biological functions through the TSLPR / ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P29/00A61P37/02A61P37/08
CPCA61P37/08C07K2317/565C07K2317/56A61K2039/505C07K16/244A61P11/06A61P11/00A61P29/00A61P37/02A61P19/02A61P37/06C07K2317/73C07K2317/24C07K2317/33C07K2317/76C07K2317/92C07K2317/55C07K16/464C07K16/465A61P37/00C07K16/2866
Inventor 王景坤
Owner CHENGDU CONMED BIOSCI CO LTD
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