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Bispecific antibody against EGFR protein and MET protein

A bispecific antibody and specific technology, applied in the field of bispecific antibody and its preparation, can solve the problems of poor guiding effect and low affinity of target molecules, etc.

Active Publication Date: 2019-07-19
北京科昕生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, bivalent bispecific or trivalent trispecific antibodies are constructed using the first constant region (CH1) of human IgG1 heavy chain and the constant region (CL) of kappa chain as the heterodimerization domain. The antigen has only one binding domain, which has a low affinity for the target molecule and poor guiding effect

Method used

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  • Bispecific antibody against EGFR protein and MET protein
  • Bispecific antibody against EGFR protein and MET protein
  • Bispecific antibody against EGFR protein and MET protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Preparation of bispecific antibodies

[0073] 1. Sequence design

[0074] The amino acid sequence composition and coding nucleic acid sequence of the antibody prepared by the present invention are shown in Table 1.

[0075] Table 1 Amino acid sequence and nucleic acid sequence of bispecific antibody

[0076]

[0077]

[0078]

[0079] 2. The expression of antibodies

[0080] 2.1 Gene synthesis and vector construction

[0081] Synthesize DNA sequences as shown in SEQ ID NO.19-35 respectively. The 5'end of the synthesized DNA sequence contains the Kozak sequence (CCCACCATGG) and the signal peptide (METDTLLLWVLLLWVPGSTG); and the 5'end contains EcoRI restriction site and 3' HindIII restriction site. Combine the synthesized DNA segment and pcDNA3.1(-) vector plasmid ( figure 1 ) Digest with EcoRI and HindIII restriction enzymes, and perform agarose gel electrophoresis to recover the corresponding digested gene fragments. Then the purified heavy and light chain coding DNA ...

Embodiment 2

[0096] Example 2 Mass spectrometric identification of bispecific antibodies

[0097] experimental method:

[0098] Take 400μg of each antibody separately, add 10μL G7PNGaseF enzyme digestion buffer, 10μL 10% NP40, 2.5μL PNGaseF, supplemented with ultrapure water, the final volume to 100μL, after mixing, wrap it with a sealing film and place it in a 37℃ water bath Warm bath overnight. The antibody sample from which the N-sugar was cut was desalted by a C4 reverse chromatographic column and analyzed by TripleTOF 5600 (AB Sciex) mass spectrometry from AB Sciex, and the data was analyzed by Analyst TF software.

[0099] result:

[0100] After purification and assembly, the antibody was modified by glycosidase to remove the N glycosyl group. The molecular weight determined by mass spectrometry is shown in Table 3. Table 3 shows that the difference between the purified and assembled antibody and the theoretical value is within the error range of the instrument, indicating that the antibody...

Embodiment 3

[0104] Example 3 Purity determination of bispecific antibodies

[0105] (1) Molecular exclusion high performance liquid chromatography

[0106] At 200mM KH 2 PO 4 , 250mM KCl, pH 6.0 mobile phase, column temperature 25℃, injection volume: 40μg, flow rate 0.6ml / min, isocratic operation for 25min, analyze bispecific antibody and control antibody samples by molecular exclusion high performance liquid chromatography The purity of the monomer and the ratio of the polymer.

[0107] (2) Sodium dodecyl sulfate capillary electrophoresis (CE-SDS)

[0108] Using sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method, under non-reducing conditions, according to the molecular weight, quantitative determination of the purity of recombinant monoclonal antibody products. Take 100μg of the sample, add 5μl of 0.8M iodoacetamide aqueous solution, vortex to mix, incubate at 70°C for 5 minutes, cool to room temperature and centrifuge at 6000 rpm for 1 minute. Take 75μl fr...

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Abstract

The invention discloses a bispecific antibody against EGFR protein and MET protein. The glycosylation modification makes the bispecific antibody contain a core fucose glycoform ratio of not more than4.5% to increase ADCC activity; and by modifying an amino acid sequence of a Fc segment, the ADCC activity of the bispecific antibody is enhanced, or the affinity for FcRn is enhanced to extend the half-life. The bispecific antibody prepared by the invention has simple preparation, stable structure and better tumor inhibition effect, and has a good market prospect.

Description

Technical field [0001] The invention belongs to the field of biomedicine and relates to a bispecific antibody and a preparation method thereof. In addition, the invention also relates to the application of the bispecific antibody. Background technique [0002] Since the first antibody drug was approved in 1986, antibody drugs have developed vigorously for more than 30 years. Because antibody drugs have obvious advantages such as high targeting, specificity, and specificity, the field of treatment has also changed from traditional cancer, Autoimmune diseases have gradually expanded to the diagnosis and treatment of anti-infection, metabolic diseases and cardiovascular diseases, and have achieved remarkable results. While improving the quality of life of patients, they also created huge economic benefits. [0003] Bispecific antibody (BsAb) is an artificial antibody containing two specific antigen binding sites, which can build a bridge between target cells and functional molecules (...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62C12N15/63A61K39/395A61P35/00
CPCA61K2039/505C07K16/2863C07K2317/31C07K2317/622C07K2319/00C07K2319/30
Inventor 王秀丽刁德坤
Owner 北京科昕生物科技有限公司
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