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PD-1 targeted polypeptide and application thereof

A technology of PD-1 and PD-L1, which is applied to peptides targeting human PD-1 protein and its application fields, and can solve the problems of easy immunogenicity, poor tissue permeability, and high production costs

Active Publication Date: 2017-10-03
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although many drugs that block the PD-1 / PD-L1 pathway have entered clinical trials or individual drugs have been approved by the US FDA, for example: nivolumab, pembrolizumab and pidilizumab targeting the PD-1 protein, and targeting and PD -L1 MPDL3280A, MDX1105, MEDI4736, etc., but most of these drugs are antibody drugs, which have the disadvantages of high production cost, poor tissue permeability, and easy immunogenicity

Method used

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  • PD-1 targeted polypeptide and application thereof
  • PD-1 targeted polypeptide and application thereof
  • PD-1 targeted polypeptide and application thereof

Examples

Experimental program
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Embodiment 1

[0084] Embodiment 1. The synthesis of polypeptide of the present invention

[0085] (1) Experimental instruments and materials

[0086] Dimethylformamide (DMF), piperidine, resin, dichloromethane (DCM), ninhydrin reagent, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate salt (HBTU), triisopropylsilane (TIS), ethanedithiol (EDT), anhydrous ether, trifluoroacetic acid (TFA), N-methylmorpholine (NMM), methanol, ethanol, 20 kinds of amino acids , peptide solid-phase synthesis tube, mass spectrometry instrument MicrOTOF-Q11 (BrukerDaltonics).

[0087] (2) Experimental steps

[0088] Weigh the resin and put it into the peptide solid-phase synthesis tube, add an appropriate amount of DMF to swell for more than half an hour. The DMF was removed, and the Fmoc deprotection reaction was carried out with a deprotection solution (piperidine:DMF=1:4), and placed on a shaking table for 10 minutes. Remove the deprotection solution, wash three times with DMF and DCM, take a s...

Embodiment 3

[0089] Example 3. Construction of Human PD-1 Recombinant Plasmid

[0090] The target gene is the coding nucleic acid of amino acids 34-150 on the PD-1 protein. The target fragment was cloned into the pET-28a vector (Novagen Company) using the specific primers designed by NcoI and NdeI restriction sites. The forward primer is: 5'-TTTTCCATGGGTCCCCCCCACCTTCTCCCCAG-3' (SEQ ID NO: 10); the reverse primer is: 5'-GCCGCCGCATATGTTATTCTGCCCTTCTCTCTG-3' (SEQ ID NO: 11). The human PD-1 gene was used as a template for PCR amplification. The PCR system is: 2 μL PD-1 plasmid, 2 μL forward primer, 2 μL reverse primer, 5 μL 10× buffer solution, 4 μL dNTP, 1 μL pfu polymerase, 34 μL ddH 2 O. PCR amplification conditions: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 48 s, 30 cycles; 10 min at 72°C. After the PCR amplification is completed, perform nucleic acid electrophoresis, cut out the target band, and recover the PCR...

Embodiment 4

[0091] Example 4. Expression and purification of human PD-1 protein

[0092] The human PD-1 protein used in the present invention is expressed and purified by prokaryotes. The specific experimental steps are as follows: the recombinant plasmid with correct sequencing was transformed into E.coli BL21 competent cells to induce expression. A single colony was picked and inoculated in TB medium containing kanamycin, and cultured overnight at 37°C with shaking. The next day, expand the small cultured product into 1L of TB medium and culture to OD 600 0.5-0.6, add the inducer IPTG to a final concentration of 0.5mM, and induce at 37°C for 5-7h. Harvest bacteria at 4,000rpm, first lyse the bacteria with lysis buffer solution (50mM Tris-HCl, pH 8.0, 50mM NaCl, 1mM DTT, 0.5mMEDTA, 5% glycerol), then high-pressure crush, and centrifuge at 12,000rpm to get the precipitate . Wash twice with washing buffer (20 mM Tris-HCl, pH 8.0, 2M urea, 2.5% Triton X-100), and collect the precipitate...

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Abstract

The invention discloses a PD-1 targeted polypeptide and an application thereof. The invention further discloses a preparation method for polypeptides and pharmaceutical compositions containing the polypeptides. The polypeptide disclosed by the invention can be bonded to a human PD-1 protein, so that the polypeptide can serve as a leader peptide for developing human PD-1 receptor inhibitors, and a foundation is provided for the development of antitumor drugs. The polypeptide disclosed by the invention can be prepared by adopting a chemical synthesis method and has the advantages of high purity, small molecular weight, safety, reliability and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention relates to polypeptides targeting human PD-1 protein and applications thereof. Background technique [0002] There are numerous co-stimulatory and co-inhibitory signals in the immune system that precisely regulate the strength and quality of T cell responses, and these inhibitory signals are called immune checkpoints. Tumor cells usually overexpress these immune checkpoint proteins, thereby inhibiting the activation of T cells and evading the killing of the immune system. Enhancing the activity of T cells through different strategies is of great significance to tumor immunotherapy, and blocking immune checkpoints is one of the effective strategies to enhance the activity of T cells. There are a series of immunosuppressive molecules on the surface of T cells, such as PD-1, CTLA-4, Tim-3, SLAM, etc. These immunosuppressive molecules can bind to their corresponding ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/11A61K38/10A61P35/00A61P35/02A61P31/04A61P31/10A61P31/12A61P29/00A61P19/08A61P19/02A61P37/02A61P7/06A61P21/04
CPCC07K7/08A61K38/00A61K38/10Y02A50/30
Inventor 李洪林刘晓峰朱丽丽李巧全丽娜吴方舒赵振江
Owner EAST CHINA UNIV OF SCI & TECH
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