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Polypeptides targeting pd-1 and applications thereof

A kind of use, amino acid technology, applied in the peptide targeting human PD-1 protein and its application field, can solve the problems of poor tissue permeability, high production cost, easy to produce immunogenicity, etc.

Active Publication Date: 2022-01-28
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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  • Polypeptides targeting pd-1 and applications thereof
  • Polypeptides targeting pd-1 and applications thereof
  • Polypeptides targeting pd-1 and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

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. Collect the bacteria at 4,000rpm, first lyse the bacteria with a 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 preci...

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Abstract

The invention discloses a polypeptide targeting PD‑1 and its application. The invention also discloses the preparation method of these polypeptides and the pharmaceutical composition containing these polypeptides. The polypeptide of the present invention can be combined with human PD-1 protein, so it can be used as a lead peptide for developing human PD-1 receptor inhibitors, and provides a basis for the development of antitumor drugs. The polypeptide of the present invention can be prepared by chemical synthesis, and has the advantages of high purity, small molecular weight, safety and reliability.

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 Patents(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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