PD-L1 targeting polypeptide as well as preparation method and application thereof

A PD-L1, targeting technology, applied in the fields of molecular biology and medicine, can solve the problems of large molecules, weak penetrating power, and poor stability in vitro

Active Publication Date: 2020-12-04
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

[0009] Antibody drugs have problems such as cumbersome preparation, poor in vitro stability, large molecules, difficu

Method used

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  • PD-L1 targeting polypeptide as well as preparation method and application thereof
  • PD-L1 targeting polypeptide as well as preparation method and application thereof
  • PD-L1 targeting polypeptide as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Construction and screening of PD-L1 targeting polypeptide library

[0075] (1) Experimental instruments and materials

[0076] 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, streptavidin Magnetic beads (MB-Streptavidin), biotin labeling kit, 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.

[0077] (2) Solvent preparation

[0078] The preparation of the deprotection solvent is hexahydropyridine: N, N dimethylformamide = 1:4;

[0079] The preparation of the reaction solution is N-methylmorpholine:N,N dimethylformamide=1:24;

[0080] The prepara...

Embodiment 2

[0106] Example 2 Detection of Affinity Between Polypeptides P1, P2, P3, P4, P5, P6 and PD-L1 Protein

[0107] The affinity between polypeptides P1, P2, P3, P4, P5 and P6 and PD-L1 protein was detected by surface plasmon resonance (SPRi), the specific method is as follows:

[0108] Spot 1mg / mL P1, P2, P3, P4, P5, P6 peptides and 1×PBS on the chip, incubate overnight at 4°C under humid conditions, then wash with 10×PBS for 10 minutes, and then wash with 1×PBS for 10 minutes , and finally washed twice with deionized water, 10min each time, immersed in 1×PBS containing 5% skimmed milk, incubated overnight at 4°C, then washed with 10×PBS for 10min, 1×PBS for 10min, and finally used Washed twice with deionized water, 10min each time, blown dry with nitrogen gas, and installed the chip on the machine (Plexera HT surface plasmon resonance imaging system).

[0109] The mobile phase was sequentially passed through 1×PBS, 2×PBS, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL and 10 μg / mL...

Embodiment 3

[0111] Example 3 Preparation of Imaging Preparations of Polypeptides P1, P2, P3, P4, P5, P6

[0112] This embodiment provides the preparation of imaging preparations for polypeptides P1, P2, P3, P4, P5, and P6, and the specific methods are as follows:

[0113] Weigh 300 mg of wang-Glu resin, cycle according to the solid-phase peptide synthesis procedure, first deprotect, and then add a certain amount of Glu, Asp, Glu, Thr, Trp, Lys, Asn and an equivalent amount of HTBU in sequence to react. After the coupling reaction, deprotect and wash. Add ε-aminocaproic acid to react, after coupling, deprotect and wash. Add FITC to carry out the coupling reaction in the dark, after the reaction, deprotect and wash. Finally, the lysate was added to the above resin to remove the side chain protecting group, and then vacuum-dried to obtain crude polypeptide fluorescent conjugates, and then purified by HPLC to obtain polypeptides P1, P2, P3, P4, P5, and P6 with a purity of 95%. An imaging p...

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Abstract

The invention relates to the technical field of molecular biology and medicine, in particular to PD-L1 targeting polypeptide as well as a preparation method and application thereof. The invention provides a PD-L1 targeting polypeptide. The general formula of the PD-L1 targeting polypeptide is X15X14X13X12X11X10X9X8X7X6X5X4X3X2X1, NX5X4X3EX2X1 or X8YX7X6TX5X4X3X2X1. The PD-L1 targeting polypeptideprovided by the invention has relatively high affinity and specificity to PD-L1, can specifically and efficiently target PD-L1 positive tumor cells, and can be used as a small molecular probe for detecting PD-L1 expression in tumor cells in practice or used as a targeting polypeptide to be conjugated or mixed with a preparation capable of killing cancer cells. The polypeptide is used for targetedtherapy and imaging of various tumors, and has a wide application prospect.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and medicine, in particular to a polypeptide targeting programmed death ligand-1 (PD-L1) and its preparation method and application. Background technique [0002] With the rapid development and mutual penetration of related disciplines such as oncology, immunology and molecular biology, tumor immunotherapy has developed into a new and important anti-tumor treatment. Different from previous surgical treatment, chemical drug treatment, radiotherapy and targeted therapy, tumor immunotherapy fights tumors by activating the body's own immune system. At present, inhibitors targeting immune checkpoints have shown good clinical effects in the treatment of various solid tumors, making people realize that immunotherapy can truly become an important tool for the treatment of malignant tumors. Tumor immunotherapy has a variety of therapeutic strategies, including non-specific immune stimulants, tu...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K7/08C12N15/11A61K47/64A61K45/00A61K49/00A61P35/00A61P19/02A61P29/00A61P31/14A61P37/08A61P7/06A61P9/10G01N33/68G01N33/574
CPCC07K7/06A61K47/64A61K45/00A61K49/0002A61P35/00A61P19/02A61P29/00A61P31/14A61P37/08A61P7/06A61P9/10G01N33/6893G01N33/57484C07K7/08Y02A50/30
Inventor 胡志远钱怡霞王蔚芝
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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