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A tumor immunotherapy predictive biomarker PD-L1 targeting polypeptide and its application

A PD-L1 and biomarker technology, applied in the field of preparation of tumor immunotherapy-related targeted peptides, can solve the problems of cumbersome preparation of antibody drugs, poor in vitro stability, weak penetration, etc., and achieve good research prospects and clinical guidance. Significance, small molecular weight, strong selective effect

Active Publication Date: 2020-08-28
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

[0011] 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 tumor immunotherapy predictive biomarker PD-L1 targeting polypeptide and its application
  • A tumor immunotherapy predictive biomarker PD-L1 targeting polypeptide and its application
  • A tumor immunotherapy predictive biomarker PD-L1 targeting polypeptide and its application

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0063] Experimental example 1 Construction and screening of the PD-L1 targeting polypeptide screening system of the present invention

[0064] 1) Experimental instruments and materials

[0065] 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, Alexa Fluor 647-anti -PD-L1 antibody, MB-Streptavidin (streptavidin magnetic beads), Streptavidin-HRP (streptavidin-labeled horseradish peroxidase), 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.

[0066] 2) Synthesis of PD-L1 "one bead, one object" polypeptide library

[0067] The Fmoc solid-phase peptide synthesis metho...

experiment example 2

[0084] Experimental example 2 Detection of affinity between PDP-4 and PDP-6 polypeptides and PD-L1 protein by surface plasmon resonance (SPRi) method

[0085] Spot 1mg / mL PDP-4 and PDP-6 polypeptides 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 use Wash twice with deionized water, 10min each time, immerse in 1×PBS containing 5% milk, incubate overnight at 4°C, then wash with 10×PBS for 10min, 1×PBS for 10min, and finally wash twice with deionized water , each time for 10min, blow dry with nitrogen, put the chip on the machine (Plexera HT surface plasmon resonance imaging system).

[0086] 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 PD-L1 to purify the protein, record and analyze SPRi signal.

[0087] As can be seen from Figure 3 (a)-(b), the SPRi signals of PDP-4 and PD...

experiment example 3

[0088] Experimental Example 3 Interaction of PDP-4 and PDP-6 with PD-L1 High Expression Cells H1975 and HCCC9810 and Normal Cell 293T

[0089] Non-small cell lung cancer cell line NCI-H1975 and cholangiocarcinoma cell line HCCC 9810 were cultured in RPMI 1640 medium containing 10% fetal bovine serum and normal human kidney fibroblast 293T were cultured in DMEM medium containing 10% fetal bovine serum , to 1 x 10 5 / mL cell concentration into a round glass-bottom Petri dish (35mm), 37 ° C, 5% CO 2After culturing in the cell incubator for 24 hours, discard the culture medium, add 1 μmol / L Hoechst 33342 and 2 μL Alexa Fluor647-anti-PD-L1 antibody to the three types of cells, incubate at 4°C for 30 minutes in the dark, and then use pre-cooled 1× After washing twice with PBS, 50 μmol / L Alexa Fluor 546-labeled PDP-4 and PDP-6 polypeptides were added, incubated at 4°C in the dark for 20 min, and then washed three times with pre-cooled 1×PBS. The fluorescence distribution in the cel...

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Abstract

The invention relates to a tumor immunotherapy prediction biomarker PD-L1 targeted polypeptide and application thereof. The general formula of the polypeptide is as follows: X1FX2X3X4X5X6X7X8KX9C. The invention further relates to a DNA fragment for encoding the polypeptide, an expression vector for expressing the polypeptide, host cells, and a bivalent, a multivalent or a pharmaceutical composition formed by the polypeptide. The preparation method is simple, the cost is low, and the practicability is high; and the tumor immunotherapy prediction biomarker PD-L1 targeted polypeptide is used for detecting the expression condition of PD-L1 in tumor cells so as to monitor the curative effect of immunotherapy in real time and screen corresponding benefited patients. The polypeptide can provide important theoretical and clinic reference basis for early diagnosis of various tumors, dynamic monitoring of examination points in immunotherapy and the like, and has important significance and high application value on optimization of the treatment scheme of individual patients.

Description

technical field [0001] The invention relates to the fields of medical biological detection technology and medicinal chemistry, in particular to the screening and application of PD-L1 tumor-specific targeting polypeptides, and in particular to a preparation method and application of tumor immunotherapy-related targeting polypeptides. Background technique [0002] In recent years, with the rapid development and mutual penetration of related disciplines such as oncology, immunology, and molecular biology, the research and development of tumor immunotherapy has been advancing by leaps and bounds. Tumor immunotherapy has become a new important anti-tumor treatment. Different from previous surgery, chemotherapy, radiotherapy and targeted therapy, tumor immunotherapy is a treatment that activates the body's own immune system to fight against tumors. It has also become a new hope for conquering malignant tumors. The core of tumor immunotherapy is to activate the anti-tumor response...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12N15/11A61K47/64A61K51/08A61K49/14A61P35/00A61P37/02
CPCA61K49/14A61K51/08C07K7/08
Inventor 王子华胡志远
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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