Multiple antibodies against human TSLP and uses thereof

A technology of monoclonal antibody and bispecific antibody, which is applied in the fields of antibodies, anti-inflammatory agents, allergic diseases, etc., and can solve problems such as the inability to control the condition of asthmatic patients

Active Publication Date: 2021-10-15
BEIJING WISDOMAB BIOTECHNOLOGY CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing therapeutic drugs include bronchodilators, glucocorticoids, combination preparations of the two (Seretide, Symbicort), leukotriene modulators, long-acting cholinergic receptor antagonists (Tiotropium bromide) , IgE antibodies, etc., cannot control the condition of all asthma patients

Method used

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  • Multiple antibodies against human TSLP and uses thereof
  • Multiple antibodies against human TSLP and uses thereof
  • Multiple antibodies against human TSLP and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0206] Example 1: Preparation of recombinant protein

[0207]The preparation of anti-TSLP antibodies requires the use of a variety of different recombinant proteins, including a long version of the human TSLP extracellular domain (hTSLP1-ECD, SEQ ID NO: 1), a short version of the human TSLP extracellular domain (hTSLP2-ECD) , SEQ ID NO:2), mouse TSLP extracellular domain (mTSLP-ECD, SEQ ID NO:3) and cynomolgus monkey TSLP extracellular domain (mfTSLP1-ECD, SEQ ID NO:4). These proteins have a large number of post-translational modifications (such as glycosylation or disulfide bonds, etc.), so the use of mammalian cell expression systems will be more conducive to maintaining the structure and function of recombinant proteins. In addition, in order to avoid the influence of the furin recognition sites in hTSLP1, hTSLP2 and mfTSLP1 on the activity of TSLP proteins, hTSLP1 mutants (hTSLP1-m, SEQ ID NO: 5) with the furin recognition sites deleted (hTSLP1-m, SEQ ID NO: 5), hTSLP2 Mu...

Embodiment 2

[0210] Example 2: Construction and screening of TSLP mouse immune library based on immobilized light chain

[0211] In order to construct a TSLP bispecific antibody based on a common light chain, the variable region of the light chain of the anti-TSLP monoclonal antibody with a specific epitope was selected to match the variable region of the mouse heavy chain that has undergone affinity maturation of the TSLP antigen in vivo, and conventional molecular biology methods were used. A single-chain antibody (scFv) library was constructed for screening anti-TSLP monoclonal antibodies against different epitopes.

[0212] After immunizing BALB / c mice aged 6-8 weeks with hTSLP1-m-His recombinant protein as immunogen, spleen cells were collected. Mouse spleen lymphocytes were isolated using mouse lymphocyte separation medium (Daktronics Biotechnology Co., Ltd., CAT#DKW33-R0100), and total cell RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., CAT#DP430), total RN...

Embodiment 3

[0214] Example 3: Identification of anti-hTSLP murine monoclonal antibodies

[0215] Using conventional molecular biology methods, nucleic acid molecules encoding the light variable region and heavy chain variable region of S17C7+L3E8-M1 were cloned into a eukaryotic expression vector to prepare and express a full antibody. At the same time, the humanized anti-TSLP monoclonal antibody Tezepelumab was prepared as a positive control with reference to US Patent US9284372B2 (the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 22; the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 23 ).

[0216] 3.1 Affinity analysis of recombinant anti-TSLP monoclonal antibody S17C7+L3E8-M1

[0217] The affinity of anti-TSLP antibodies was determined by surface plasmon resonance using a Biacore X100. Amino Coupling Kit (BR-1000-50), Human Antibody Capture Kit (BR-1008-39), CM5 Chip (BR100012) and 10×HBS-EP (BR100669) with...

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Abstract

The invention provides a bispecific antibody against human thymic stromal lymphopoietin (TSLP), a monoclonal antibody and uses of the bispecific antibody and the monoclonal antibody.

Description

[0001] Field of Invention [0002] The present invention generally relates to the fields of genetic engineering and antibody drugs; in particular, the present application relates to bispecific antibodies against human TSLP, monoclonal antibodies and uses thereof. [0003] Background of the Invention [0004] Human thymic stromal lymphopoietin (TSLP) is also known as IL-7-like cytokine 1 , is a member of the IL-2 cytokine family. TSLP is mainly secreted by epithelial cells of the thymus, lung, gut and skin 2,3 , followed by some fibroblasts, airway smooth muscle endothelial cells, mast cells, monocytes, granulocytes, dendritic cells (DC) 4-14 . The expression of TSLP is regulated by some factors, such as allergens, pro-inflammatory factors (IL-1β, TNF-α, TGF-β, IL-4, IL-13), Th2 factors, trauma, mechanical damage, bacteria, etc. 15-18 . [0005] The target cells of TSLP co-express TSLP receptor (Thymic stromal lymphopoietin receptor, TSLPR) and IL-7 receptor alpha chain (IL...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46A61K39/395A61P11/06A61P37/02A61P29/00A61P19/02A61P9/14A61P37/08A61P11/00A61P17/00A61P11/02A61P17/06A61P13/12A61P1/00A61P25/00
CPCC07K16/2866A61P11/06A61P37/02A61P29/00A61P19/02A61P9/14A61P37/08A61P11/00A61P17/00A61P11/02A61P17/06A61P13/12A61P1/00A61P25/00C07K2317/31C07K2317/565C07K2317/56A61K2039/505
Inventor 张雪萍刘志刚周晓巍胡俊杰郝小勃刘玉兰
Owner BEIJING WISDOMAB BIOTECHNOLOGY CO LTD
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