Two kinds of anti-her2 antibody-chaetokin conjugates and their preparation method and anti-tumor application

A technology of chaetin and chaetin, which is applied in the field of biotechnology and medicine, to reduce toxic and side effects, have great therapeutic application value, and enhance the effect of killing

Inactive Publication Date: 2021-04-13
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the candidate drug warheads in the clinic are highly concentrated on dolastatin, maytansine and other drugs. Finding new drug warheads is of great significance to the development of ADCs drugs, and marine natural products are an important source of anti-tumor active substances. High research and development value, and there are no reports on the coupling of Trastuzumab and Pertuzumab to other marine toxins for the treatment of HER2-mediated diseases

Method used

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  • Two kinds of anti-her2 antibody-chaetokin conjugates and their preparation method and anti-tumor application
  • Two kinds of anti-her2 antibody-chaetokin conjugates and their preparation method and anti-tumor application
  • Two kinds of anti-her2 antibody-chaetokin conjugates and their preparation method and anti-tumor application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: Preparation of vc-Chaetocin drug molecule with linker

[0050] The preparation steps are as follows:

[0051] (1) Mix chaetocin (+)-Chaetocin 15mg (0.022mmol) with N,N-diisopropylethylamine 16.5μL (0.100mmol), 2.4mL N,N-dimethylformamide at room temperature under nitrogen After 20 min of magnetic stirring under protection, take 37 mg (0.050 mmol) of maleimide-modified valine-citrulline dipeptide and 17 μL (0.103 mmol) of N,N-diisopropylethylamine into the reaction solution , stirred overnight at room temperature;

[0052] (2) TLC monitors the completion of the reaction, quenches the reaction solution with 0.40mL 1.3M trifluoroacetic acid and 0.60mL acetic acid, concentrates by rotary evaporation with an oil pump, and purifies by silica gel column chromatography. The eluent is dichloromethane:methanol=6:1 , to obtain 6.8 mg of brown-yellow solid product 6-L-C, yield: 24.4%. 1 H NMR (500MHz, CD 3 OD)δ H (ppm): 8.08 (d, J=6.0Hz, 2H, -CH=CH-), 7.57-7.30 (...

Embodiment 2

[0054] Example 2: Preparation of Anti-HER2 Antibody-Chaitin Conjugate (T-L-C or P-L-C)

[0055] The preparation method of T-L-C conjugate comprises:

[0056] (1) Replace the trastuzumab antibody solution (20 mg / mL) with PBS reaction buffer (pH 7.4) through a Sephadex G-25 desalting column, and use an ultrafiltration tube to concentrate the monoclonal antibody to adjust the concentration to 10 mg / mL;

[0057] (2) Take 500 μL (10 mg / mL) trastuzumab solution, add 21 μL of 20 mM TCEP solution with a 12-fold excess molar ratio, and incubate at 37 ° C for 1.5 h;

[0058] (3) Use PBS containing 1mM DTPA to pass through a desalting column to purify the reduced monoclonal antibody, add 64 μL of 5.38mM 6-L-C solution with a 10-fold excess molar ratio to the monoclonal antibody solution under ice bath conditions, and let it stand for 1.5h ;

[0059] (4) Add 35 μL of 20 mM cysteine ​​solution with a 20-fold excess molar ratio, let stand for 45 minutes, pass PBS through a desalting colum...

Embodiment 3

[0071] Embodiment 3: in vitro cell proliferation assay biological activity experiment

[0072] In this example, the effects of chaetocin (+)-Chaetocin on the proliferation of various tumor cell lines were studied; with HER2 positive cells SKBR-3, AU565, HER2 negative cells MBA-MD-231 and normal cells HaCaT as objects, The biological activities of Trastuzumab, Pertuzumab monoclonal antibody and T-L-C, P-L-C conjugates were determined.

[0073] In this example, the CCK-8 reagent was used to evaluate the anti-proliferation effect of the drug. The main component of the reagent is water-soluble tetrazolium salt WST-8. WST-8 is bioreduced by intracellular dehydrogenase to generate orange-yellow water-soluble formazan (formazan). The amount of formazan produced is linearly related to the number of living cells. .

[0074] The cell lines selected in this example are: gastric cancer cell SGC-7901, lung cancer cell NCI-H460, liver cancer cell SMMC-7721, breast cancer cell SKBR-3, AU56...

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Abstract

The present invention relates to the fields of biotechnology and medicine, specifically two anti-HER2 antibody-chaetocin conjugates Trastuzumab-vc-Chaetocin (T-L-C) and Pertuzumab-vc-Chaetocin (P-L-C) and their Synthetic preparation methods and applications. The antibody conjugate is obtained by conjugating a drug molecule with a linker and a monoclonal antibody, and its structure is shown below. The invention also provides a preparation method of the drug molecule with linking group. The antibody conjugate prepared by the present invention has a targeting effect on HER2-positive cells, which can not only enhance the killing of HER2-positive tumor cells, but also reduce the toxic and side effects caused by (+)-Chaetocin administered alone. Cell-induced cancers have enormous potential therapeutic applications.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to two anti-HER2 antibody-chaetokin conjugates, a preparation method thereof and anti-tumor application. Background technique [0002] Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family with tyrosine kinase activity. Polymerization of receptors leads to phosphorylation of receptor tyrosine residues and initiates multiple signaling pathways leading to cell proliferation and tumorigenesis, and is highly expressed mainly in breast cancer. The overexpression of HER2 gene is not only related to the occurrence and development of tumors, but also an important clinical treatment monitoring and prognostic indicator, and an important target for the selection of tumor targeted therapy drugs. The research on this target is relatively mature, and Herceptin (Trastuzumab), a drug against the overexpression of this gene, has been av...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/395A61K31/548A61K47/68A61K47/65A61P35/00C07K16/32
CPCA61K31/548A61K39/3955C07K16/32A61K2300/00
Inventor 王钰洁曹鑫卢小玲刘小宇焦炳华
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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