Anti-aggregation humanized IgG (immunoglobulin G) antibody CH2 structural domain mutant and application

An anti-aggregation and structural domain technology, applied in the direction of antibody mimics/scaffolds, anti-animal/human immunoglobulins, hybrid peptides, etc., can solve the problems of reduced drug efficacy, weak anti-aggregation ability, and poor stability, and achieve Good stability, good anti-aggregation ability, and the effect of reducing the risk of clinical use

Active Publication Date: 2018-06-22
武汉班科生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, antibodies developed with wild-type CH2 fragments have their own limitations, such as poor stability, weak anti-aggregation ability, and tend to aggregate or degrade during the process of expression, purification, storage, etc.
The immunogenicity caused by protein aggregation not only reduces the efficacy of the drug, but also brings great risks to clinical application

Method used

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  • Anti-aggregation humanized IgG (immunoglobulin G) antibody CH2 structural domain mutant and application
  • Anti-aggregation humanized IgG (immunoglobulin G) antibody CH2 structural domain mutant and application
  • Anti-aggregation humanized IgG (immunoglobulin G) antibody CH2 structural domain mutant and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Amino acid sequence and nucleotide sequence of m01sm1

[0019] The m01sm1 skeleton provided by the present invention is obtained by the following method: firstly, a phage display library for random mutation of amino acids in the aggregation-prone region of m01s is constructed. Then, the phage display library was screened with specific anti-CH2 antibody to obtain candidate clones. Then it was sent for sequencing, and m01sm1 was obtained after identification.

[0020] The amino acid sequence of m01sm1 is Seq ID No.1, and the coding gene sequence is Seq ID No.2.

Embodiment 2

[0021] Example 2: Molecular conformation and existence form (monomer, dimer, etc.) of m01sm1

[0022] AKTA analysis of the existing form of m01sm1: Concentrate the purified m01sm1 protein to 1mg / ml, use PBS (pH7.4) as the elution buffer, pass through Column Superdex75Increase 10 / 300GL, and the flow rate is 1ml / min, and then determine the existing form testing, the result is figure 1 , compared with the standard curve, it can be found that the molecular weight of the protein is about 14kDa, and the result shows that they exist in the form of monomers.

[0023] CD detection of protein molecular conformation: Dilute the m01sm1 protein to 0.3 mg / ml, PBS (pH7.4) as a control, and detect its average molar ellipticity under different wavelength conditions at the near ultraviolet end (wavelength λ=190nm~260nm), such as figure 2 , there is an obvious trough at λ=218nm, indicating that the secondary structure of the above protein is β-sheet.

Embodiment 3

[0024] Example 3: Comparison of m01s and m01sm1 anti-aggregation

[0025] Turbidity experiment: Adjust the final protein concentration of m01s and m01sm1 to 1mg / ml, treat them in a water bath at 50°C, take samples at regular intervals, detect the absorbance at λ=320nm and record the data, among them, the PBS solution is used as a blank control, and the comparison The strength of anti-aggregation with m01s protein, from image 3 In A, it can be found that the m01sm1 signal rises slowly, indicating that its anti-aggregation ability is better.

[0026] ThT fluorescence experiment: adjust the final concentration of m01s and m01sm1 protein to 1mg / ml, add ThT, use 440nm as excitation light and 482nm as absorption light, and measure the fluorescence change within 250min. By measuring the luminescence intensity of m01sm1 and m01s proteins combined with ThT at different times, the anti-aggregation properties of m01sm1 and m01s proteins were compared, from image 3 In B, it can be fou...

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Abstract

The invention discloses an anti-aggregation humanized IgG (immunoglobulin G) antibody CH2 structural domain mutant and an application. A CH2 fragment of an IgG antibody serves as an object; an easy aggregation area is predicted on a basis of the existing transformation framework m01s; and further optimization is performed to form a new framework m01sm1. M01sm1 has higher stability than the mutantm01s; the development of a more stable C single domain antibody is facilitated; production, purification and storage cost of a monoclonal antibody or Fc fusion protein can be lowered; the anti-aggregation ability of m01sm1 is better; and clinical use risks caused by protein aggregation can be reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-aggregation human IgG antibody CH2 domain mutant and its application. Background technique [0002] With its strong specificity and high sensitivity, antibodies are widely used in various fields, especially in life science research and clinical treatment such as ELISA, Western blot, immunofluorescence analysis, rapid diagnosis of diseases, and as biological agents to treat various diseases. Therefore, since entering the 21st century, people have begun to pay more and more attention to the research and development and clinical application of therapeutic antibody drugs, because it has less toxic and side effects on the human body, has natural and highly specific curative effects, and has created a huge social benefits and economic benefits. At the same time, the miniaturized antibody developed based on the CH2 fragment has good tissue penetration due to its relatively small mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K19/00
CPCC07K16/18C07K2317/524C07K2319/30
Inventor 龚睿
Owner 武汉班科生物技术有限公司
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