Anti-IL-1beta monoclonal antibody and application thereof

A monoclonal antibody and sequence technology, applied in applications, antibodies, anti-inflammatory agents, etc., can solve the problems of reduced gout attack rate and high incidence of adverse reactions

Active Publication Date: 2017-07-07
CHANGCHUN GENESCIENCE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with colchicine, canakinumab reduces gout attacks by 48%-75%. Compared with triamcinolone acetonide, it can effe...

Method used

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  • Anti-IL-1beta monoclonal antibody and application thereof
  • Anti-IL-1beta monoclonal antibody and application thereof
  • Anti-IL-1beta monoclonal antibody and application thereof

Examples

Experimental program
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preparation example Construction

[0056] The specific anti-IL-1β monoclonal antibody preparation and related detection methods are as follows: Construction of IL-1β fully human antibody library, including optimization of phage display vectors, isolation of PBMC cells, RNA extraction, cDNA reverse transcription and antibody genes Amplification and enzyme digestion, ligation, electroporation, etc.; screening of antibodies, including antigen preparation, phage display screening, ELISA screening, etc.; construction of full-length antibodies, expression, purification, and affinity determination, cell activity determination, etc.

[0057] The anti-IL-1β monoclonal antibody provided by the present invention has a heavy chain variable region and a light chain variable region:

[0058] (I) The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID No: 1-6; or one or more amino acids are substituted, deleted or added; or the amino acid sequence shown in (I) Amino acid sequences with at leas...

Embodiment 1

[0154] The preparation of embodiment 1 antigenic protein

[0155] 1. Antigen and positive antibody gene synthesis and expression vector construction:

[0156] The amino acid sequence design of fusion human IL-1β mature protein amino acid sequence and connecting peptide-mIgG1Fc is shown in SEQ ID NO:25. The amino acid sequence design of fusion human IL-1β mature protein amino acid sequence and connecting peptide-6his is shown in SEQ ID NO:26.

[0157] The amino acid sequence corresponding to the human IL-1β mature protein fusion protein designed above (IL-1β-linked peptide-mIgG1Fc or IL-1β-linked peptide-6his) is artificially optimized for codons, and the codon-optimized sequence is shown as SEQ ID NO: 27 or 28 add Hind III restriction site and Kozak sequence GCCGCCACC at the 5' end, add stop codon TAG and EcoR I restriction site at the 3' end, and entrust Suzhou Synbio Company to synthesize the optimized DNA , cloned into pUC57simple (provided by Suzhou Synbio) vector to obt...

Embodiment 2

[0169] Example 2: Construction of natural human single chain antibody phage display library

[0170] 1. Construction of phagemid vector

[0171] Select pCANTAB5E as the phage display vector, and carry out vector transformation according to the needs of cloning and phage display. The transformation results are as follows: figure 2 . The SfiI-NcoI-XhoI+Linker+NheI-NotI sequence (SEQ ID NO: 31) was gene-synthesized, then digested with SfiI and NotI, and ligated with the pCANTAB5E vector for recombinant construction to obtain the transformed vector pCANTAB5E-SF.

[0172] 2. PBMC isolation and mRNA extraction

[0173] Aseptically extract fresh peripheral blood from healthy volunteers, use lymphocyte separation medium (GE) to separate the lymphocytes therein, and use Invitrogen’s reagent (15596-026) extracts 100×10 6 The total RNA of each cell, the result is as follows image 3 shown.

[0174] 3. Antibody library primer design, synthesis and RT-PCR

[0175] According to th...

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Abstract

The invention provides the technical field of antibody medicine, discloses a fully-humanized human anti-IL-1beta monoclonal antibody screened through the phage antibody library technology and prepared through the genetic engineering technology, and discloses a DNA molecule for encoding the monoclonal antibody, a DNA molecule expression carrier for encoding the monoclonal antibody, a host cell and application. The anti-IL-1beta monoclonal antibody can prevent bonding of human IL-1beta and human IL-1R. By means of bonding of IL-1beta, blocking of IL-1beta and a receptor signal channel of the IL-1beta, unnecessary IL-1beta initial induced heat transfer, amplified lymphocyte responses and stimulation acute stage responses can be reduced. The anti-IL-1beta monoclonal antibody can be used for detecting IL-1beta expressions and used for prevention and treatment of cryopyrin-associated periodic syndromes.

Description

technical field [0001] The invention relates to the technical field of antibody drugs, in particular to a fully human anti-IL-1β monoclonal antibody and its application. Background technique [0002] Interleukin-1β (IL-1β) is an important cytokine and polypeptide regulatory factor mainly produced by monocyte-macrophages, which plays a regulatory role in the activation of cellular immunity. IL-1β contains 153 amino acids, and its theoretical molecular weight is 17.5KD. IL-1β, does not contain disulfide bonds, but two cysteine ​​(Cys) residues are required for IL-1β activity. [0003] IL-1β has strong biological activities, including: (1) mediating inflammatory response: IL-1β can not only cause inflammatory response itself, but also induce cyclooxygenase 2 (cyclooxygenase-2, COX-2), iNOS , IL-6 and other inflammatory factors, thereby further activating stromal cells and immune cells to produce more IL-1β to participate in the inflammatory response; (2) immune regulation, co...

Claims

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Application Information

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IPC IPC(8): C07K16/24C12N15/13C07K14/545A61K39/395A61P29/00G01N33/68G01N33/577
CPCA61K2039/505C07K14/545C07K16/005C07K16/245C07K2317/56C07K2317/76C07K2317/92
Inventor 冯晓王涛金磊秦锁富陈宇珩
Owner CHANGCHUN GENESCIENCE PHARM CO LTD
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