Binding protein for targeted binding of HER2 and preparation method and application of binding protein

A protein-binding and targeted binding technology, which is applied in the field of immunology, can solve the problems of poor thermal stability, complex preparation process, and large molecular weight of probes, and achieve the effects of low cost, low non-specific binding rate, and small molecular weight

Inactive Publication Date: 2020-10-30
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, radionuclide labeling of monoclonal antibodies, such as 89 Although Zr-trastuzumab can be combined with HER2, its slow clearance in blood, low tissue penetration, large molecular weight and complex structure have also led to problems such as poor thermal stability and complicated preparation process of such probes.

Method used

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  • Binding protein for targeted binding of HER2 and preparation method and application of binding protein
  • Binding protein for targeted binding of HER2 and preparation method and application of binding protein
  • Binding protein for targeted binding of HER2 and preparation method and application of binding protein

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preparation example Construction

[0049] The present invention also provides a method for preparing a binding protein targeting HER2, the preparation method comprising the following steps:

[0050] preparing a DNA molecule encoding the binding protein;

[0051] Prepare the expression vector of the above-mentioned DNA molecule;

[0052] introducing the expression vector into a host cell; and

[0053] Express target binding protein.

[0054] The present invention also provides the use of the prepared binding protein targeting HER2 in the preparation of drugs or reagents for diagnosing or treating tumors, wherein the drugs or reagents are molecular imaging probes, and the molecular imaging probes The imaging preparation includes any one of radionuclide, radionuclide label or molecular imaging preparation; the tumor includes early breast cancer, metastatic breast cancer or metastatic gastric cancer and HER2-positive tumors.

Embodiment 1

[0057] Example 1 is a preparation method for targeting binding to HER2 binding protein (BindHer).

[0058] 1. Gene synthesis and cloning construction of BindHer molecules.

[0059] Use protein design software to design a protein (BindHer) with a target binding to HER2, and perform gene synthesis according to its amino acid sequence. Cysteine ​​was introduced at the carboxyl terminus of BindHer to facilitate radionuclide labeling. In order to facilitate expression, the BindHer plasmid was subcloned into the pET29a vector to construct the pET29a-BindHer plasmid. The plasmid was digested and verified and sequenced with an automatic sequencer. After verifying that the vector was successfully constructed, it was transformed into an expression strain of E.coli BL21(DE3).

[0060] 2. Expression and purification of recombinant protein.

[0061]Insert the monoclonal bacterium of E.coliBL21(DE3) Escherichia coli containing the pET29a-BindHer expression plasmid into the LB liquid medi...

Embodiment 2

[0065] In Example 2, the affinity of BindHer to HER2 protein was detected.

[0066] at Biacore TM Surface plasmon resonance was used to analyze the interaction between BindHer protein and HER2 on the T200 (GE Healthcare, USA) system.

[0067] 1) Recombinant HER2 extracellular domain (HER2-ECD) was immobilized on the surface of CM5 chip (amine coupling method). HER2-ECD protein (10004-HCCH, Sino Biological Inc., Beijing China) was coupled to the amine of the carboxylated dextran layer on the surface of the CM5 (BR-1000-12, GE Healthcare, USA) sensor chip.

[0068] a) Dextran surface activation: 0.4M N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide) and 0.1M N-hydroxy (N-hydroxysuccinimide) was mixed at a ratio of 1:1 (vol / vol), and the flow rate was 10 μL / min for 10 minutes.

[0069] b) Binding to HER2-ECD: Dissolve 50 μg / mL of HER2-ECD in 10 mM acetic acid, pH 4.5 buffer, and run at a flow rate of 10 μL / min for 5 minutes.

[0...

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Abstract

The invention provides a binding protein for targeted binding of HER2 and a preparation method and application of the binding protein. The binding protein comprises the following amino acid sequence (a): NDEMRX1TYW X2IALF X3 X4L X5N X6X7KR X8 X9IR X10LYDDP X11X12A X13 X14LEX15 X16A X17LEA X18 X19 X20. The binding protein disclosed by the invention can be combined with HER2 and has good affinity with HER2. A radionuclide molecular imaging probe can be prepared through nuclide labeling and used for molecular imaging diagnosis, tumor imaging diagnosis can be improved to the molecular level of tumor cell specific expression, and the HER2 expression situation of possible tumor lesions in vivo can be monitored in real time before a treatment scheme is determined or in the process of monitoring drug treatment.

Description

technical field [0001] The invention relates to the technical field of immunology, and in particular to a binding protein targeting HER2, its preparation method and application. Background technique [0002] Individualized precise treatment of tumor targeted therapy drugs is an important issue worthy of attention in the field of cancer diagnosis and treatment. Human epidermal growth factor receptor-2 (Human epithelial growth Factor receptor-2, HER2) is closely related to cell growth, activation and sensitivity to radiotherapy and chemotherapy. HER2 monoclonal antibody therapy, represented by Trastuzumab and Pertuzumab, has significantly improved the prognosis of HER2-positive breast cancer patients, and is an important milestone in tumor molecular targeted therapy. Detecting the expression of HER2 and monitoring HER2 during the course of the disease is of great significance for targeted therapy. Detecting the expression of HER2 in primary and metastatic lesions is the key t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K19/00C12N15/11C12N1/21C12N15/70A61K51/08A61K101/02A61K103/10A61K103/00C12R1/19
CPCC07K14/00C12N15/70A61K51/08C07K2319/00
Inventor 李炯黄浓郁曹洋魏于全张阳
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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