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A polypeptide targeting tumor stem cell marker cd133 and its application

A tumor stem cell and marker technology, used in anti-tumor drugs, medical preparations with non-active ingredients, peptides, etc., can solve the problems of difficult labeling, weak penetration, large molecules, etc. No immunogenicity, small molecular weight effect

Active Publication Date: 2020-05-19
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the cumbersome preparation of antibody drugs, poor in vitro stability, large molecules, difficult labeling, weak penetration, post-translational modification and high cost, the further application of antibody drugs is limited.

Method used

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  • A polypeptide targeting tumor stem cell marker cd133 and its application
  • A polypeptide targeting tumor stem cell marker cd133 and its application
  • A polypeptide targeting tumor stem cell marker cd133 and its application

Examples

Experimental program
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experiment example 1

[0056] Experimental example 1 Construction and screening of CD133 targeting polypeptide screening system of the present invention

[0057] 1) Experimental instruments and materials

[0058] N-methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), ninhydrin, vitamin C, phenol, tetramethyluronium hexafluorophosphate (HBTU), hexahydro Pyridine, triisopropylsilane (TIS), ethanedithiol (EDT), N,N dimethylformamide (DMF), anhydrous ether, resin, methanol, various Fmoc protected amino acids, tetraphenylethylene ( TPE), PE-anti-CD133 antibody, MB-Streptavidin (streptavidin magnetic beads), peptide synthesis tube, shaker, vacuum water pump, rotary evaporator, laser confocal microscope (ZEISS LSM 710), the above reagents and Materials were obtained from commercial sources.

[0059] 2) Synthesis of CD133 "one bead one object" polypeptide library

[0060]Use the Fmoc solid-phase peptide synthesis method to synthesize the peptide library, the specific met...

experiment example 2

[0077] Experimental example 2 Detection of affinity between CS133-P2 polypeptide and CD133 protein by surface plasmon resonance (SPRi) method

[0078] Spot the 1mg / mL CS133-P2 polypeptide and 1×PBS on the chip, incubate overnight at 4°C under humid conditions, then wash with 10×PBS for 10 minutes, then wash with 1×PBS for 10 minutes, and finally wash with deionized water for 2 minutes. Once, 10min each time, immersed in 1×PBS containing 5% milk, incubated overnight at 4°C, then washed with 10×PBS for 10min, 1×PBS for 10min, and finally washed twice with deionized water, 10min each time , blown dry with nitrogen, and put the chip on the machine (Plexera HT surface plasmon resonance imaging system).

[0079] The mobile phase was sequentially passed through 1×PBS, 2×PBS, 0.78μg / mL, 1.56μg / mL, 3.125μg / mL, 6.25μg / mL, 12.5μg / mL and 25μg / mL human CD133 purified protein, and the SPRi signal was recorded and analyzed .

[0080] Depend on figure 2 It can be seen that the SPRi sign...

experiment example 3

[0081] Experimental Example 3 CS133-P2 and CD133 isolated from leukemia + Specific staining of tumor stem cells and tissue sections of colorectal cancer patients

[0082] CD133 was sorted by CD133 antibody flow cytometry + Tumor stem cells were cultured in complete stem cell medium containing cell growth factors for 24 hours at 37°C, and then mixed with 1×10 3 / mL cell concentration into a round glass-bottomed culture dish (35mm), add 1μmol / L Hoechst 33342 and 5μL PE-anti-CD133 antibody to the three kinds of cells respectively, incubate at 4°C for 60min in the dark, then use pre-cooled 1 Wash twice with ×PBS, add 50 μmol / L FITC-labeled CS133-P2 polypeptide respectively, incubate at 4°C in the dark for 20 min, and wash three times with pre-cooled 1×PBS. Fluorescence distribution in stem cells was detected with a laser scanning confocal microscope (ZEISS LSM 710). Tumor tissue samples were fixed with paraformaldehyde for more than 48 hours (paraformaldehyde was replaced once ...

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Abstract

The invention relates to a tumor stem cell marker CD133-targeting polypeptide and an application thereof. A general formula of the polypeptide is CX1X2X3X4X5X6X7X8LX9. The invention further relates to a DNA fragment encoding the polypeptide, an expression vector and host cell expressing the polypeptide, and a bivalent body, a multivalent body or a pharmaceutical composition formed by the polypeptide. The polypeptide is used for detecting and targeting the tumor stem cell, and is good in specificity and sensitivity, simple in preparation method, low in cost and high in practicability. The polypeptide is applied to near-infrared fluorescence imaging to carry out high-sensitivity and high-resolution monitoring and targeted therapy on the CD133 tumor stem cell in a skin transplatation tumor. Simple, fast, economical and accurate detection means can be provided for real-time monitoring of peripheral blood CD133 expression of a patient and the correlation of the CD133 tumor stem cell in tumor recurrence, metastasis, drug tolerance and prognosis, so that the therapeutic scheme is convenient to timely adjust and novel means can be provided for improving the prognosis of patients.

Description

technical field [0001] The invention relates to the fields of biomedical detection and medicinal chemistry, in particular to the screening of tumor stem cell CD133-specific targeting polypeptide and its application as a drug carrier. Background technique [0002] Malignant tumors are one of the diseases that seriously threaten human life. In recent decades, the morbidity and mortality of tumors have been increasing worldwide. For most malignant tumors, surgery, chemotherapy, radiation, targeted and immunotherapy can be used to kill most tumor cells, but the vast majority of cancer patients still cannot be cured, and are prone to recurrence and metastasis. The survival time of malignant tumor patients and quality of life are consistently lower. In 2001, the concept of cancer stem cell (CSC) was formally proposed. The cancer stem cell (CSC) hypothesis believes that there are a very small number of tumor cells in tumor tissue that act as stem cells. They have the characterist...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K14/00A61K47/42A61K47/66A61P35/00A61P37/02A61P35/02
CPCA61K47/42C07K7/06C07K14/00
Inventor 王子华王蔚芝胡志远
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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