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HER2 linear epitope neutralizing active monoclonal antibody and its application

A monoclonal antibody, linear table technology, applied in the field of immunology, to achieve the effect of enhancing the therapeutic effect and inhibiting the proliferative growth

Active Publication Date: 2020-01-10
北京天成新脉生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports of monoclonal antibody drugs targeting Domain 2 / 3 sites

Method used

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  • HER2 linear epitope neutralizing active monoclonal antibody and its application
  • HER2 linear epitope neutralizing active monoclonal antibody and its application
  • HER2 linear epitope neutralizing active monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 produces the establishment of the hybridoma cell line of clone2H4 and clone2D9

[0034] 1. Experimental materials

[0035] 1. Immunogen: HER2 is used as the target protein, and a polypeptide sequence (peptide at amino acid positions 266-296 of Domain 2 in the extracellular region of HER2) is selected from it. The sequence is as follows: SLAFLPESFDGDPASNTAPLQPEQLQ.

[0036] The above-mentioned polypeptide is prepared by chemical synthesis, and the purity requirement is greater than 90%. The polypeptide is coupled with KLH to prepare an immunogen.

[0037] 2. Culture medium: DMEM medium was purchased from Hyclone Company, HAT, HT selection medium, and pristane were purchased from Sigma Company.

[0038] 3. Experimental animals: Balb / c mice, 8-12 weeks old, female, SPF grade animal culture.

[0039] 4. Other materials: Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma Company, PEG4000 was purchased from Fluka Company, HRP...

Embodiment 2

[0065] The preparation of the monoclonal antibody of embodiment 2 anti-HER2

[0066] 1. Antibody preparation

[0067] Adult BALB / c mice were selected, and pristane was inoculated intraperitoneally, 0.5ml per mouse. After 7-10 days, the 16th generation hybridoma cells were inoculated intraperitoneally, 1×10 per mouse 6 -2×10 6 indivual. After an interval of 5 days, when the abdomen is obviously enlarged and the skin feels tense when touched with hands, the ascites can be collected with a No. 9 needle.

[0068] Centrifuge the ascitic fluid (13000r / min for 30 minutes), remove cell components and other precipitates, and collect the supernatant. Purify with Protein G~Sepharose CL-4B, the upper column solution is 20mM PBS buffer solution, and the eluent of column chromatography is: pH2.7, 20mM glycine buffer solution to obtain the monoclonal antibody against HER2.

[0069] 2. Antibody Identification

[0070] 1. Antibody purity identification:

[0071] SDS-PAGE electrophoresis...

Embodiment 3

[0078] Example 3 Affinity Experiment of Purified Antibody

[0079] 1. Cell ELISA method

[0080] The clone2H4 and clone2D9 monoclonal antibodies were used for cell affinity test to determine their binding EC50 value to HER2-positive SK-BR-3 cells, as shown in Table 3.

[0081] table 3

[0082]

[0083] Detection method: Prepare SK-BR-3 cells on the first day, select well-growing SK-BR-3 cells, wash three times with sterile PBS, add 0.1% trypsin to digest the cells for 1-3 minutes, add 10 5ml of DMEM medium with %FBS, cells were obtained after centrifugation, and adjusted to 1×10 after cell counting 5 / ml, spread 200μl per well into 96 wells, and culture overnight. On the second day, after the culture supernatant was removed from the cells, the cells were washed twice with PBS, and fixed with 95% alcohol for 15 min. After the cells were fixed, the cells were washed twice with sterile water, and then 200 μl / well of 5% skimmed milk was added to block for 1 h. After washin...

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Abstract

The invention provides an HER2 linear epitope monoclonal antibody with a neutralization activity and applications thereof. Polypeptides, which comprise amino acids between 374 to 398th in the HER2 extracellular region Domain 2 / 3 and have a sequence represented by SEQ ID No.5, and carrier protein coupled immunized mice are used to prepare multiple strains of hybridomas, which excretes an monoclonal antibody, which has a neutralization activity and is capable of specifically recognizing breast cancer cell surface HER2. The invention provides applications of the polypeptides in the preparation of HER2 monoclonal and drugs for disease treatments taking HER2 as the target. The affinity of the combination between the monoclonal antibody and HER2 is in a range of 1 to 4 nM. By combining the monoclonal to the HER2 on the surface of tumor cells, the proliferation of tumor cells can be effectively inhibited, the endocytosis of HER2 is induced, the monoclonal antibody is an ideal antibody for biological targeting treatments, the provided monoclonal body can be used with trastuzumab / pertuzumab, the curative effect is enhanced, and the market value of the monoclonal antibody is obvious.

Description

technical field [0001] The invention relates to the field of immunology and antibody preparation technology, in particular to anti-HER2 linear epitope neutralizing active monoclonal antibody and application thereof. Background technique [0002] Human epidermal growth factor receptor 2 (HER2), a member of the epidermal growth factor receptor (ErbB2) family, is a transmembrane receptor-like protein with a relative molecular mass of 185,000 and has tyrosine kinase activity. HER2 is an important prognostic factor for breast cancer. HER2-positive (overexpressed or amplified) breast cancer has special clinical characteristics and biological behaviors, and its treatment mode is also very different from other types of breast cancer. Drugs acting on the HER-2 target are currently the most commonly used and most effective drugs for the treatment of breast cancer, mainly including trastuzumab and pertuzumab. [0003] Genentech launched Herceptin (trastuzumab for injection), the first...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N5/20A61K39/395A61K39/00A61P35/00G01N33/577
Inventor 孙乐李茂华张翠娟张小刚王慕旸
Owner 北京天成新脉生物技术有限公司
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