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Development and application of a therapeutic agent for TSLP-related disorders

A technology of use and sequence, applied in the field of antibodies recognizing TSLP protein

Active Publication Date: 2022-07-15
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 a therapeutic agent for TSLP-related disorders
  • Development and application of a therapeutic agent for TSLP-related disorders
  • Development and application of a therapeutic agent for TSLP-related disorders

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0179] Example 1. Preparation of TSLP and TSLPR recombinant proteins

[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 Sinobiological, Beijing, catalog number CG90911 -UT), the signal peptide MDMRVPAQLLGLLLLWLRGARS was inserted into the N-terminus, and a tag containing 6 histidines was fused and expressed at the carboxyl-terminus of TSLP to obtain pSect-hTSLP-cHis and pSect-cyTSLP-cHis plasmids, and this plasmid was transfected into HEK293. 6E cells (ATCC CRL-1573), the cell supernatant was collected, but no recombinant protein of corresponding length was obtained by nickel column affinity chromatography. Then the full-length fragment of TSLP was cloned into the prokaryotic expression plasmid pET-30a, and BL21 E.coli. was transformed with this plasmid. After inducing expression, it was found tha...

Embodiment 2

[0183] Example 2. Construction of TSLPR stably transfected cells

[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 by the Lenti-Pac HIV lentiviral packaging kit (Guangzhou Funeng Gene Co., Ltd.), and transfected Ba / F3 cells were screened for resistance by adding puromycin to obtain a cell line Ba / F3-hTSLPR that can stably express human TSLPR. Figure 4 showed that the constructed cells were confirmed to express human TSLPR by detecting the binding of biotinylated human TSLP protein to cell surface TSLPR, and its EC 50 =0.22nM.

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

[0187] IL-7Rα is involved in the signal...

Embodiment 3

[0191] Embodiment 3, animal immunity

[0192] 6-week-old female Balb / C mice were selected for immunization. Each mouse was immunized with 50 μg of antigen each time. The antigen was alternately immunized with human and cynomolgus monkey TSLP. The antigen was mixed with equal volume of Freund's adjuvant for subcutaneous immunization, every two weeks. Immunity once. After four immunizations, tail blood was collected, and ELISA was used to detect the titer of mouse serum and the inhibitory effect of serum on the binding of TSLP to TSLPR. Or after the initial immunization, booster immunization was performed every other week by using a gene gun, mixed with gold powder with human TSLP expression plasmid, and bombarded with bare skin on the abdomen of mice at a pressure of 400 psi, for a total of nine immunizations.

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Abstract

The present disclosure is the development and application of a therapeutic agent for TSLP-related disorders, and relates to an antibody or an antigen-binding portion thereof that binds to a TSLP protein, and a manufacturing method and application thereof. The antibody can bind to human TSLP and / or cynomolgus monkey TSLP with high affinity, block the binding of TSLP to TSLPR, and inhibit the transduction of TSLP-stimulated signals through the STAT5 pathway.

Description

technical field [0001] The present disclosure relates to an antibody that recognizes TSLP protein, and a method for making and using the same. Background technique [0002] Thymic Stromal Lymphopoietin (TSLP) is a cytokine of the interleukin-7 family, mainly produced by epithelial cells and keratinocytes located in the skin, intestine or lung, and is involved in maintaining the mucosal immune system. Stablize. After epithelial tissue is stimulated or destroyed by allergens or pathogens, dendritic cells present antigens to naive CD4+ T cells, while releasing epithelial cytokines, TSLP and IL-33. These important co-stimulatory 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 exerts biological functions through the TSLPR / IL-7Rα receptors on the surf...

Claims

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Application Information

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Patent Type & Authority Patents(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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