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Anti-human Delta-like4 humanized antibody and preparation and application thereof

A technology of humanized antibodies and antibody fragments, applied in the field of bioengineering, can solve the problem of low affinity and specificity of mouse antibodies

Active Publication Date: 2016-03-09
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the antibodies screened by phage antibody library technology are fully human, their affinity and specificity are still not high compared to murine antibodies

Method used

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  • Anti-human Delta-like4 humanized antibody and preparation and application thereof
  • Anti-human Delta-like4 humanized antibody and preparation and application thereof
  • Anti-human Delta-like4 humanized antibody and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Application of bioinformatics software to simulate the three-dimensional structure of mouse monoclonal antibody Fv region ( figure 1 ), and analyze the classic amino acids necessary to maintain the conformation of the CDR region according to its three-dimensional structure, search the human Fabsequence database according to the classic amino acids in the FR region, and find the most suitable human FR template, such as figure 2 As shown, Z27506 and J00256 are the human FR templates of the heavy chain variable region, and X63399 and J00242 are the human FR templates of the light chain variable region. Replace the FR region of the mouse monoclonal antibody with a human template to obtain a CDR-grafted antibody (VH g / VL g ), on this basis, the important classic amino acids in the FR region were selected for back mutation, and the heavy and light chain variable regions of the humanized antibody H3L2 (expressed as VH 3 , VL 2 ).

[0034] The resulting humanized heavy c...

Embodiment 2

[0037] Electroporation and co-transfection of CHO-S cells were carried out with the above-mentioned four successfully constructed vector plasmids, and the electroporation transfection conditions were: 160V, 15ms. After 24 hours, add G418 for selection (final concentration is 700-1000 μg / ml), change the medium every 2-3 days, and clones grow after about 10 days, pick clones and culture them in 96-well plates, and take the supernatant after the cells are full. For DotBlot, the working antibody is HRP-labeled goat anti-human IgG (H+L). According to the DotBlot results, select positive clones and expand them into 24-well plates for culture, then take the supernatant for DotBlot; select positive clones and expand them into T-25 culture flasks for culture, take the supernatant for WesternBlot, select cell lines with correct expression and assembly, freeze them and store them. The second round of screening was carried out, and the screening steps were the same as above. After two ro...

Embodiment 3

[0040] The cell culture supernatant was suction-filtered with a 0.22 μm filter membrane, and then loaded into the Protein A chromatography column through a peristaltic pump. After loading the sample, the column was washed with Bindingbuffer until the UV signal line became straight. The sample was then eluted with 0.1 M pH 3.0 sodium acetate buffer (Elutionbuffer) for 5 column volumes. In order to neutralize the acidic substances in the Elutionbuffer, add 1MpH9.0Tris to the sample receiving tube at 100μl / ml eluent; the eluted sample is analyzed for SDS-PAGE purity and replaced by ultrafiltration, and the Bio-Tek protein microquantifier is used to analyze the Antibody concentration was measured.

[0041] see results Figure 4 .

[0042] Example 4 SPR determination of antigen-antibody affinity.

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Abstract

The present invention provides anti-human DLL4 (delta-like 4) humanized monoclonal antibody H3L2 and a preparation method and application thereof. A CDR-grafting-based FR region important base reversible mutant humanized antibody of a murine monoclonal antibody is designed by use of bioinformatics software, the heavy chain variable region of the anti-human DLL4 humanized antibody has an amino acid sequence shown as SEQ IDNO: 3, the light chain variable region of the anti-human DLL4 humanized antibody has an amino acid sequence shown as SEQ IDNO: 4; and the humanized antibody H3L2 is capable of high affinity binding to human DLL4 antigen (KD value of 2.26* 10<-12>M), can specifically target HUVEC cell surface DLL4 antigen, blocks DLL4 inhibition effect on HUVEC cell proliferation, and retains the biological activity of the parent murine monoclonal antibody. An antibody-based treatment method for diagnosis and treatment of diseases characterized by DLL4 overexpression is provided.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to an anti-human Delta-like4 humanized antibody H3L2 and its preparation and application. Background technique [0002] signal path [0003] The Notch pathway is a conserved signal transduction pathway, which plays an important role in the regulation of cell development, growth and apoptosis during the early development of various tissues and organs. Cancer stem cell biology studies have found that the Notch signaling pathway plays a key role in tumor stem cells maintaining tumor growth, and abnormal activation of the Notch signaling pathway has been found in many blood cancers and solid tumors. Vascular endothelial cells express two Notch receptors (Notch-1 and Notch-4), and all ligands except DLL-3. Among them, DLL4 is a specific ligand of endothelial cells, and the Notch-1 signaling pathway mediated by it plays an important role in angiogenesis and development. Many a...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/85C12N5/10A61K39/395A61P35/00
CPCC07K16/28C07K2317/24
Inventor 王旻吴旻贾雪莲许卓斌王世静王泽根张娟罗晨
Owner CHINA PHARM UNIV
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