Application of anti-EGFR scFv:: FTH1/FTH1 protein nanoparticles

A nanoparticle and protein technology, applied in the field of nanobiomedicine, can solve the problem of lack of specific targeting of triple-negative breast cancer cells, and achieve the effect of inhibiting proliferation and survival, inhibiting effect, and high safety

Pending Publication Date: 2021-06-18
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The problem to be solved by the present invention is to overcome the deficiency in the prior art of molecularly targeted drugs that specifically target triple-negative breast cancer cells, inhibit tumor growth, and have low toxic and side effects, thereby providing an anti-EGFR scFv::FTH1 / Application of FTH1 protein nanoparticles in the preparation of anti-triple-negative breast cancer preparations, and application of pharmaceutical compositions or kits containing anti-EGFR scFv::FTH1 / FTH1 protein nanoparticles in the preparation of anti-triple-negative breast cancer products

Method used

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  • Application of anti-EGFR scFv:: FTH1/FTH1 protein nanoparticles
  • Application of anti-EGFR scFv:: FTH1/FTH1 protein nanoparticles
  • Application of anti-EGFR scFv:: FTH1/FTH1 protein nanoparticles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation and Characterization of Anti-EGFR ScFv::FTH1 / FTH1 Protein Nanoparticles

[0035] Two plasmids, pET-28(+)-FTHI and pET-28a(+)-anti-EGFR scFV-FTH1, exist in our laboratory (see ZL201010239499.3 for specific preparation methods). Wherein, the amino acid sequence of the anti-EGFR scFv::FTH1 protein is shown in SEQ ID NO:1, and the amino acid sequence of the FTH1 protein is shown in SEQ ID NO:2. Escherichia coli competent cells E.coli.BL21(DE3) and E.coli.DH5a were purchased from Beijing Tiangen Biochemical Technology Company. The above two plasmids were transformed into E.coli.DH5a competent cells, and the positive clones were screened by colony PCR and enzyme digestion identification. Afterwards, the expression vector with the correct sequence was transformed into Ecoil.BL.21(DE3) competent cells to obtain engineering expression strain cells containing FTH1 protein gene target engineering expression strain cells and anti-EGFR scFv::FTH1 protein gene ...

Embodiment 2

[0041] Example 2 Safety evaluation of anti-EGFR scFv::FTH1 / FTH1 protein nanoparticles in vivo

[0042] Reagent preparation:

[0043] 4% Paraformaldehyde: Dissolve 40g of paraformaldehyde in 1000mL of PBS, and filter through a 0.22μm membrane filter.

[0044] Table 1.1 Experimental grouping

[0045]

[0046]

[0047] experimental method:

[0048] As shown in Table 1.1, healthy Balb / c mice (Shanghai Experimental Animal Center, Chinese Academy of Sciences (Shanghai Slack Experimental Animal Co., Ltd.)) were randomly divided into 3 groups, 3 mice in each group, injected intraperitoneally, 1 time / 2 days , administered 3 times in a row. On the 7th day, the whole blood of the mouse was collected by picking the eyeball to contain EDTA-K 2 in the anticoagulant tube. Then measure the blood indexes of mice, the main indexes include: white blood cell number (WBCs), lymphocyte number (Lymphs), monocyte number (Mons), neutrophil number (Grans), red blood cell number (RBCs), plate...

Embodiment 3

[0077] Example 3 Anti-EGFR scFv:: Effect of FTH1 / FTH1 Protein Nanoparticles on the Survival Rate of EGFR Positive Cells

[0078] Cell line:

[0079] Human triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 (Shanghai Experimental Animal Center, Chinese Academy of Sciences (Shanghai Slack Experimental Animal Co., Ltd.)). Its complete medium is: L-15 medium containing 10% fetal bovine serum, culture conditions: 100% air, no CO 2 , 37 ℃ constant temperature.

[0080] Reagent preparation:

[0081] 5mg / mL MTT solution: Weigh 250mg MTT and dissolve it in 50mL PBS, dissolve it fully at 60°C, filter it with a 0.22μm sterile filter membrane, pH 7.4, and store it in the dark at -20°C.

[0082] Determination of cell viability by MTT assay

[0083] experimental method:

[0084] MDA-MB-231 and MDA-MB-468 cells grown on the adherent wall were digested with trypsin and counted, respectively in 2 × 10 3 and 5×10 3 Cells were seeded in a 96-well plate at a density of 100 ...

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Abstract

The invention discloses application of an anti-EGFR (epidermal growth factor receptor) scFv:: FTH1/FTH1 protein nanoparticle in preparation of a preparation for resisting triple negative breast cancer. The anti-EGFR scFv:: FTH1/FTH1 protein is formed by compounding an anti-EGFR scFv:: FTH1 protein and an FTH1 protein. Compared with commercial anti-EGFR monoclonal cetuximab, EGFR and a signal channel thereof can be better down-regulated under the condition of the same administration dosage, and a more remarkable inhibition effect on proliferation of tumor cells expressing EGFR and growth of tumors of tumor-bearing mice is achieved, so that a better anti-tumor effect is shown, the cetuximab has relatively high safety in a living body, and the anti-EGFR scFv:: FTH1/FTH1 protein provides a wide prospect for the application of EGFR target-based protein nano-drugs.

Description

technical field [0001] The invention belongs to the field of nano-biomedicine, and relates to the application of an anti-EGFR scFv::FTH1 / FTH1 protein nanoparticle, in particular to an anti-EGFR scFv::FTH1 / FTH1 protein nanoparticle in the preparation of anti-triple-negative breast cancer preparations Applications. Background technique [0002] Breast cancer (Breast Cancer, BC) is one of the most common malignant tumors and the leading cause of cancer-related deaths among women worldwide. Triple-negative breast cancer (TNBC) accounts for approximately 10-20% of all breast malignancies, and this histologic subtype lacks expression of progesterone receptor (PR) and estrogen receptor (ER), and also does not express human epidermal Growth factor 2 (HER2). Given the lack of these targeted receptors, chemotherapy remains the standard treatment for TNBC, with very limited other treatment options. Among breast cancer patients, TNBC patients usually have a poor prognosis because it ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K39/395A61P35/00
CPCC07K16/2863C07K14/47A61P35/00C07K2319/00C07K2317/622A61K2039/505
Inventor 曹旭妮李子慧陈慧
Owner EAST CHINA UNIV OF SCI & TECH
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