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Anti-il-1β humanized monoclonal antibody and preparation method and application thereof

An antibody and variable region technology, applied in the field of anti-IL-1β humanized monoclonal antibody and its preparation, can solve the problems of short half-life, strong immunogenicity, and low specificity, and achieve improved affinity and strong IL Effect of -1β neutralizing ability and high antibody affinity

Active Publication Date: 2019-08-30
SHANGHAI CHEMPARTNER CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the strong immunogenicity and short half-life of the existing anti-IL-1β non-humanized monoclonal antibody, but the low affinity and low specificity of the anti-IL-1β humanized monoclonal antibody Strong defects, providing a high-affinity and specificity anti-IL-1β humanized monoclonal antibody and its preparation method and application

Method used

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  • Anti-il-1β humanized monoclonal antibody and preparation method and application thereof
  • Anti-il-1β humanized monoclonal antibody and preparation method and application thereof
  • Anti-il-1β humanized monoclonal antibody and preparation method and application thereof

Examples

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Effect test

Embodiment 1

[0035] Example 1 Template single-chain antibody gene synthesis and CDR mutant phage library construction

[0036] The sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the template antibody are as follows figure 1 As shown, the GGGGSGGGGSGGGGS polypeptide sequence is linked between VH and VL to form a single-chain antibody scFv, and the corresponding DNA sequence can be obtained by full-length gene synthesis. Perform detailed CDR site analysis on the VH and VL sequences of the template antibody, and design random mutation primers for CDR-H2, CDR-H3, CDR-L1, and CDR-L3 regions accordingly. Using mutation primers to do PCR synthesis of VH and VL gene fragments containing mutations, and then performing PCR splicing into new mutant scFv single-chain antibody genes. The mutated scFv gene was digested and ligated into the phage display vector pCANTAB5E (GE Healthcare Life Sciences), which was then used to transform TG1 Escherichia coli cells ...

Embodiment 2

[0037] Example 2 Affinity-based screening of phage antibody mutant library

[0038] The constructed phage mutant library was used to pan against IL-1β antigen. Phage panning was performed in liquid phase using biotinylated antigen. At lower and lower antigen concentrations, only phages displaying high-affinity antibodies will bind to the antigen, which can then be called out and retained using streptavidin magnetic beads. After several times of panning, the library was sampled and sequenced. There was no significant enrichment of new variants in the CDR-H3 and CDR-L3 libraries, while most clones in the CDR-H2 and CDR-L1 libraries had been converted to new variants. variant.

[0039] 50 different clones were selected from the CDR-H2 and CDR-L1 libraries after panning, and scFv proteins were induced to express and purified. Then use BiacoreT200 to quickly detect its dissociation curve with the antigen and compare the Off-rate. Among them, clones that dissociate slowly from t...

Embodiment 3

[0040] Example 3 Expression, purification and affinity constant determination of full-length antibody IgG in eukaryotic cells

[0041] Using the obtained single-chain antibody gene containing the "AI" mutation as a template, the human antibody heavy chain gene was spliced ​​with the human full-length antibody IgG heavy chain constant region by overlapping PCR, and then digested with HindIII and EcoRI restriction enzymes Afterwards, it was purified and recovered by agarose gel electrophoresis, and then ligated into the pcDNA3.1(+) plasmid (Invitrogen Company) to construct the human heavy chain eukaryotic expression vector pcDNA3.1(+)(VHCH). Similarly, using the single-chain antibody gene containing the "SVV" or "RTV" mutation as a template, the human antibody light chain gene was spliced ​​with the human full-length antibody IgG light chain constant region by overlapping PCR, and then HindIII and EcoR I were used to After digestion with restriction enzymes, it was purified and ...

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Abstract

The invention discloses an anti-IL-1beta humanized monoclonal antibody, a preparation method and application thereof. The anti-IL-1beta humanized monoclonal antibody is an anti-IL-1beta single-chain antibody, full-length IgG antibody or Fab antibody, and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is a peptide chain formed by substitution of one amino acid on one or two of the 57th amino acid and the 60th amino acid in a peptide chain of the amino acid sequence shown as SEQ ID No:1 in a sequence table, and / or, the light chain variable region is a peptide chain formed by formed by substitution of one amino acid on one or more of the 27th amino acid, the 28th amino acid and the 29th amino acid in a peptide chain of the amino acid sequence shown as SEQ ID No:2 in the sequence table. Compared with the existing anti-IL-1beta antibodies, the antibody provided by the invention has higher affinity and stronger neutralizing ability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-IL-1β humanized monoclonal antibody and its preparation method and application. Background technique [0002] IL-1β, namely interleukin 1β, is an important member of the IL-1 family, its precursor protein has a molecular weight of 31KD, and the mature IL-1β has a molecular weight of 17KD. IL-1β is mainly involved in the process of inflammatory response, autoimmune diseases and leukemia, and it also has a direct impact on vascular endothelial cells, macrophages, T cells and B cells. IL-1β has been implicated in inflammation in many pathological conditions, including infection (viral, bacterial, fungal and parasitic), endotoxic shock, arthritis, rheumatoid arthritis, pelvic inflammatory disease, multiple sclerosis, asthma, osteoarthritis Arthritis, psoriasis, Alzheimer's disease, Crohn's disease, Peyronies' disease, heart disease (eg, atherosclerosis), colon cancer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/24C12N15/13C12N15/70C12N7/01C12N15/85C12N5/10C12P21/02A61K39/395
Inventor 闫树德吴辰冰
Owner SHANGHAI CHEMPARTNER CO LTD
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