Human thymus stromal lymphopoietin monoclonal antibody and application thereof

A lymphocyte and progenitor technology, applied in the direction of antibodies, anti-cytokine/lymphokine/interferon immunoglobulin, microorganisms, etc., can solve the problems affecting the quality of life, health care burden, etc.

Active Publication Date: 2020-05-26
IPHASE THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most patients with moderate and severe asthma remain symptomatic or poorly controlled, affecting quality of life and having a significant health care burden

Method used

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  • Human thymus stromal lymphopoietin monoclonal antibody and application thereof
  • Human thymus stromal lymphopoietin monoclonal antibody and application thereof
  • Human thymus stromal lymphopoietin monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0160] The preparation of embodiment 1 immunogen

[0161] The eukaryotic expression vector of human TSLP was constructed, transfected into 293F cells, and the culture supernatant was purified by Protein A column to prepare human TSLP protein, which was used for mouse immunization, clone screening and functional identification.

[0162] (1) Construction of human TSLP eukaryotic expression vector

[0163] The human source TSLP sequence starts from Tyr 29 of natural human TSLP to Gln 159, a total of 131 amino acids (sequence shown in SEQ ID NO.102), and a mouse Fc fragment (mFc) (Accession number: AJ487681, Allele or Gene:IGHG1*01), can be combined with the recombinant protein A in the protein A column, thereby can be purified by affinity chromatography, therefore, construct rhTSLP-mFc fusion protein (amino acid sequence such as SEQ ID NO.103 shown).

[0164] The above rhTSLP-mFc fusion gene was amplified by PCR, and the recombinant plasmid was constructed by homologous recombi...

Embodiment 2

[0170] Example 2 Mouse immunization and antibody titer determination in serum

[0171] Use rhTSLP-His (rhTSLP coupled with a His tag, the sequence is shown in SEQ ID NO.104) or rhTSLP-mFc prepared in Example 1 as an immunogen for Balb / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Ltd) for immunization. A total of 10 mice were immunized, among which 6 mice were immunogened with rhTSLP-His and immunized with complete Freund's adjuvant; the other 4 mice were immunogened with rhTSLP-mFc and quickly immunized with water-soluble adjuvant.

[0172] When using complete Freund's adjuvant for immunization, multi-point subcutaneous injection was used on the back, and the immunization dose was 50 μg / 200 μl / mouse. Mix 50 μg rhTSLP-His with 100 μl Freund’s complete adjuvant (Sigma, F5881) for the first immunization, mix 50 μg rhTSLP-His with 100 μl Freund’s adjuvant for the second immunization, third immunization, fourth immunization and fifth immunization Co...

Embodiment 3

[0179] The preparation of embodiment 3 hybridomas

[0180] After the last immunization in Example 2, the spleens of the two mice (No. 1 and No. 6 immunized with complete Freund's adjuvant; No. 2 and No. 4 rapidly immunized) with the highest titers obtained by each immunization method were mixed respectively, After milling in normal saline, the suspension rich in B cells was taken, and cell fusion was carried out with myeloma cell SP2 / 0 under the action of fusion agent PEG (Sigma, P7181). The fused cells were divided into 15 96-well cell culture plates, placed in 5% CO in 20% fetal bovine serum RPMI-1640 full medium (Thermo, 31800089) containing HAT (Sigma, H0262) 2 After one week of culture at 37°C, HT medium (Sigma, H0137) was used to continue the culture. Screening and subcloning of embodiment 4 hybridomas

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Abstract

The invention relates to the technical field of antibodies, and especially relates to a human thymus stromal lymphopoietin monoclonal antibody and an application thereof. The TSLP antibody can be specifically combined with hTSLP, has high affinity, and can effectively inhibit the combination of TSLP and a receptor thereof so as to inhibit activation of a downstream signal path and activation of immune cells by TSLP. The TSLP antibody has great significance in detection of the content of hTSLP and allergic asthma, and diagnosis, treatment and prognosis of inflammatory or allergic inflammatory diseases such as chronic obstructive pulmonary disease, allergic dermatitis, allergic rhinitis, allergic nasosinusitis, eosinophilic granulocytic esophagitis, allergic conjunctivitis, inflammatory bowel disease or atopic dermatitis.

Description

technical field [0001] The invention relates to the technical field of antibodies, in particular to a human thymus stromal lymphopoietin monoclonal antibody and an application thereof. Background technique [0002] Thymic stromal lymphopoietin (TSLP) is produced by epithelial cells, airway smooth muscle cells, lung fibroblasts, and stromal cells in the thymus, lung, skin, intestine, and tonsils after exposure to allergens, viruses, bacteria, cigarettes, and environmental Particle post-induced trigger generation. Human thymic stromal lymphopoietin (hTSLP) is an interleukin 7 (IL-7)-like cytokine. The mature hTSLP consists of 131 amino acid residues and presents a typical 4-strand α-helix bundle structure. [0003] The TSLP receptor complex is a heterodimer composed of TSLP receptor (TSLPR) and IL-7 receptor alpha (IL-7Ra), mainly in dendritic cells, CD4 + Expressed on T cells, eosinophils, basophils, mast cells and type 2 innate lymphoid cells (ILC2). The cells with the hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24C12N5/20A61K39/395A61P35/00A61P37/08A61P29/00G01N33/68
CPCA61P29/00A61P35/00A61P37/08C07K16/244C07K2317/24C07K2317/56C07K2317/565C07K2317/567C07K2317/76C07K2317/92G01N33/6869G01N33/6893G01N2333/5418G01N2800/52
Inventor 刘鸿君李耀东姜风超宗娟娟
Owner IPHASE THERAPEUTICS LTD
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