Fucose-removed anti-HER2 antibody and application thereof

A fucose and fucoid-removing technology, applied in the field of fucose-removing anti-HER2 antibodies, can solve the problem that patients cannot benefit from Herceptin, and achieve inhibition of tumor angiogenesis, high ADCC activity, and the probability of drug resistance small effect

Inactive Publication Date: 2017-03-29
BIORAY LABORATORIES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In other cancers, HER2 is overexpressed in about 43-69%, but in general, HER2 is moderately or lowly expressed in most tumors, although drugs targeting HER2 are considered an established treatment for HER2 Drugs for positive tumors, currently most patients cannot benefit from Herceptin

Method used

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  • Fucose-removed anti-HER2 antibody and application thereof
  • Fucose-removed anti-HER2 antibody and application thereof
  • Fucose-removed anti-HER2 antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Expression and purification of antibodies

[0077] The antibody light and heavy chain sequences were cloned into mammalian expression vectors using the human growth factor 1 (hEF) promoter. The expression vector was transfected into CHO-S cells or FUT-8 knockout CHO-S cells, the cells were grown in Freestyle Max reagent (purchased from Invitrogen) medium, and stable expression strains were screened with puromycin. The stable cell line was cultured in a 1-liter shake flask, and the supernatant was collected after 14 days, and the antibody was purified using a protein A column. After the antibody was eluted from the column, it was dialyzed with PBS to obtain the purified antibody. If the antibody is from CHO-S cells, a high-fucose anti-HER2 antibody will be obtained; if the antibody is from a FUT-8 gene knockout CHO-S cell, a fucose-free anti-HER2 antibody will be obtained. There are three kinds of DUXB11, DG44 and CHOK1 in CHO cells, among which CHOK1 is wild...

Embodiment 2

[0078] Example 2: Comparison of fucose content in fucose-free anti-HER2 antibody and high-fucose anti-HER2 antibody.

[0079] Antibody glycans were purified from 100 μg of antibody after digestion with trypsin and glycopeptidase, followed by mass spectrometry analysis. The identification of each glycan depends on its mass-to-charge ratio m / z, and all detected glycan concentrations are derived from comparison with an internal reference.

[0080] Glycosyl nomenclature

[0081]

[0082] ◆ N-acetylneuraminic acid N-acetylgalactosamine glucose

[0083] Galactose N-Glucosamine N-glycolylgalactosamine

[0084] ● Mannose Fucose

[0085] Glycosyl number definition: 5 numbers respectively express the number of different sugars, six-carbon sugar (Galactose, Mannose, or Glucose), N-acetylhexosamine (GlcNAc or GalNAc), fucose (Fucose, FUC for short), N-acetylneuraminic acid (Neu5Ac), and N-glycolylneuraminic acid (Neu5Gc). The third number represents whether fucose i...

Embodiment 3

[0096] Example 3: Comparison of the affinities of the anti-HER2 antibody with no fucose and the anti-HER2 antibody with high fucose to the ECD of the extracellular region of HER2.

[0097] The experiments were performed on a Biacore T200. The mobile phase is PBS buffer (NaCl 8.0g, KCl 0.2g, KH 2 PO 4 0.24g, Na 2 HPO 4 ×12H 2 O 3.628g, be dissolved in 800ml distilled water, adjust pH value to be 7.4 with hydrochloric acid, distilled water is settled to 1000ml), all samples are all dissolved in the above-mentioned PBS damping fluid.

[0098] The fucose-free anti-HER2 antibody and the high-fucose anti-HER2 antibody were immobilized on the CM5 sensor chip through amino coupling, and the flow rate of the flow cell was set to 10ul / min, and the concentration of 0.1mol / L 1-B The surface of the CM5 chip was activated with an equal volume mixed solution of 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.1mol / L N-N-hydroxysuccinimide (NHS). Inject 50mg / ml of fucose-...

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Abstract

The invention provides a fucose-removed anti-HER2 antibody which is produced by cells with the FUT8 gene knocked out. The amino acid sequence of the fucose-removed anti-HER2 antibody is shown as SEQ ID NO: 1, and the nucleotide sequence is shown as SEQ ID NO: 2. Furthermore, the antibody comprises a CH2 structural domain; the amount of fucose in the CH2 structural domain is zero. The invention further provides a medicine with the fucose-removed anti-HER2 antibody serving as an active ingredient, and a reagent, a composition or a kit with the fucose-removed anti-HER2 antibody serving as an active ingredient. Compared with a fucose-containing anti-HER2 antibody, the antibody disclosed by the invention is higher in activity, and shows good tumor inhibition activity in both expression and low expression of herceptin-resistant HER2.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fucose-free anti-HER2 antibody and its application. Background technique [0002] According to the report of the World Health Organization, global cancer cases will show a rapid growth trend, from 14 million in 2012 to 19 million in 2025, and will reach 24 million in 2035. Developing countries in Africa, Asia, and Central and South America have the most severe cancer incidence. In 2012, there were 14 million new cancer cases and 8.2 million deaths worldwide. [0003] The morbidity and mortality of cancer in China have been increasing, and since 2010 it has become the leading cause of death and a major public health problem in China. According to the 2015 China Cancer Statistics Report, there are expected to be 4,292,000 new cancer cases and 2,814,000 cancer deaths in 2015, with lung cancer having the highest morbidity and mortality. The morbidity and mortality of gas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/32C12N15/13A61K39/395A61P35/00
CPCC07K16/32A61K2039/505C07K16/2863C07K2317/732C07K2317/76C07K2317/92
Inventor 侯理理刘明耀余波
Owner BIORAY LABORATORIES INC
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