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Anti-human IL-4-RA antibody and uses thereof

An antibody and antigen technology, applied in the direction of antibody, anti-animal/human immunoglobulin, application, etc., can solve the problem of low affinity

Active Publication Date: 2020-09-11
AKESO BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently existing anti-human IL-4RA antibody drugs have low affinity, and there is still a lack of an anti-human IL-4RA antibody with high affinity

Method used

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  • Anti-human IL-4-RA antibody and uses thereof
  • Anti-human IL-4-RA antibody and uses thereof
  • Anti-human IL-4-RA antibody and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0330] Example 1 Preparation of anti-human IL-4RA antibodies 20G10, 13E5 and 18H7

[0331] 1 Preparation of hybridoma cell lines 20G10, 13E5 and 18H7

[0332] The antigen IL-4RA-mFc used to prepare the anti-IL-4RA antibody is a fusion protein of human IL-4RA mature peptide (GenbankID: NP_001244336.1) and mFc tag (SEQ ID NO: 121) (synthesized by Zhongshan Kangfang Biopharmaceutical Co., Ltd. ) and used to immunize BALB / C mice (purchased from Guangdong Medical Experimental Animal Center). With reference to existing technical methods (for example, Stewart, S.J., "Monoclonal Antibody Production", in Basic Methods in Antibody Production and Characterization, Eds.G.C.Howard and D.R.Bethell, Boca Raton: CRCPress, 2000), BALB / C Spleen cells of mice (purchased from Guangdong Medical Experimental Animal Center) and mouse myeloma cells formed hybridoma cells by cell fusion technology. Use IL4RA-hFc protein (where IL-4RA is the same as above, hFc is human IgG Fc purification tag, specif...

Embodiment 2

[0338] Example 2: Sequence analysis of antibody 13E5 against human IL-4RA

[0339] The mRNA was extracted from the LT008 cell line cultured in Example 1 according to the method of Cultured Cell Bacteria Total RNA Extraction Kit (Tiangen, Cat. No. DP430).

[0340] According to Invitrogen III First-Strand Synthesis System for RT-PCR Kit Instructions Synthesize cDNA and perform PCR amplification.

[0341] The PCR amplification product was directly cloned by TA, and the specific operation was performed referring to the instructions of the pEASY-T1 Cloning Kit (TransgenCT101) kit.

[0342] The products of TA clones were directly sequenced, and the sequencing results were as follows:

[0343] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the fragment length is 348bp. The encoded amino acid sequence is shown in SEQ ID NO: 2, with a length of 116 amino acids, wherein the sequence of heavy chain CDR1 is shown in SEQ ID NO: 9, and the seq...

Embodiment 3

[0345] Example 3: Design and preparation of humanized antibodies 13E5 H1L1, 13E5 H2L2, 13E5 H3L3, 13E5 H4L2 and 13E5 H4L4 against human IL-4RA

[0346] 1. Design of light chain and heavy chain sequences of anti-IL4RA humanized antibodies 13E5 H1L1, 13E5 H2L2, 13E5 H3L3, 13E5 H4L2 and 13E5 H4L4

[0347] According to the three-dimensional crystal structure of human IL-4RA protein (Hage T, Reinemer P, Sebald W. Crystals of a 1:1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain. Eur J Biochem.1998; 258 (2) : 831-6.) and the sequence of the antibody 13E5 obtained in Example 2, through computer simulation of the antibody model, design mutations according to the model, to obtain the variable region sequences of the antibodies 13E5 H1L1, 13E5H2L2, 13E5 H3L3, 13E5 H4L2 and 13E5 H4L4 (antibody The constant region sequence is from the NCBI database, Ig gamma-4 chain C region, ACCESSION: P01861.1; the light chain constant region is Ig kappa chai...

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Abstract

The invention relates to an anti-human interleukin-4-receptor A antibody, a pharmaceutical composition or test kit containing the same and application thereof.

Description

technical field [0001] The invention belongs to the fields of allergic disease treatment and molecular immunology, and relates to an anti-human interleukin 4-receptor A antibody, a pharmaceutical composition or kit containing it, and uses thereof. Background technique [0002] Interleukin-4 receptor (IL-4R) is a transmembrane receptor with two different forms: one is type I IL-4R, and the high-affinity IL-4R d subunit (ie IL-4RA in the present invention), and moderate affinity γC subunit, it combines interleukin-4 (interleukin-4, IL-4) and mediates biological functions such as cell proliferation and activation induced by IL-4; The other is type II IL-4R, which is composed of high-affinity IL-4Rα subunit (IL-4RA) and interleukin-13 receptor α subunit (IL-13Rα). The cognate functional receptor of (interleukin-13, IL-13) can bind IL-13 (Wang and Secombes, Cytokine. 2015. Volume 75, Issue 1, Pages 8-13 (2015)). IL-4R is expressed in T, B, hematopoietic stem cells, endothelial ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N5/20A61K39/395A61P37/08A61P11/02A61P35/00A61P37/02A61P17/00A61P11/00A61P11/06A61P29/00G01N33/68G01N33/577G01N33/574
CPCC07K16/2866A61P37/08A61P11/02A61P35/00A61P37/02A61P17/00A61P11/00A61P11/06A61P29/00C07K2317/565C07K2317/56C07K2317/567C07K2317/92C07K2317/24C07K2317/76C07K2317/74A61K2039/505G01N33/6893C07K2317/31G01N2333/7155G01N2800/20C07K2317/55
Inventor 李百勇夏瑜王忠民张鹏
Owner AKESO BIOPHARMA
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