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PD-1 targeted blocking peptide and application thereof

A PD-1, targeting technology, applied in the biological field to achieve anti-tumor effect, significant effect, and the effect of restoring lymphocyte activity

Active Publication Date: 2020-05-29
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the characteristics of the PD-1 / PD-L1 interaction, that is, the surface-surface interaction interface of the protein is relatively flat, the range of action sites is large and highly variable, so it is becoming a challenge to find small molecule inhibitors with blocking effects. a challenging job

Method used

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  • PD-1 targeted blocking peptide and application thereof
  • PD-1 targeted blocking peptide and application thereof
  • PD-1 targeted blocking peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Preparation and activation of host bacteria:

[0032] The following operations need to be performed aseptically in an ultra-clean workbench:

[0033] 1. Melt 100mL LB solid medium, add 100μL tetracycline stock solution when the temperature drops to 50-60℃, mix well and pour about 20mL into a sterile petri dish (Φ=0.9cm) to prepare a final concentration of 20μg / mL Tetracycline resistance-LB plate (Tet-LB plate);

[0034] 2. Streak inoculate the strain E.coli ER2738 stored at -70°C on a Tet-LB plate, culture it upside down at 37°C overnight (about 16-18h), and store it in a refrigerator at 4°C for two weeks;

[0035] 3. Prepare a sterile test tube containing 2 mL of LB liquid medium, add 2 μL of tetracycline stock solution with a concentration of 20 mg / mL, and the final concentration is 20 μg / mL;

[0036] 4. Pick the ER2738 monoclonal colonies on the Tet-LB plate and put them into 2 mL of Tet-LB medium, and cultivate overnight at 37°C with shaking.

Embodiment 2

[0038] Amplification of phage library:

[0039] 1. Inoculate 200 μL of overnight cultured ER2738 into 200 mL of sterilized LB liquid medium, and culture with vigorous shaking at 37°C for about 2 to 2.5 hours, so that it reaches the logarithmic growth phase (OD600 nm absorbance value is 0.4 to 0.6);

[0040] 2. Take 1.5 μL of the original phage library and dilute it in 150 μL of TBS buffer solution; infect 100 μL of the diluted phage library with 200 mL of ER2738 bacteria solution in logarithmic phase;

[0041]3. Mix well and let stand for 15 minutes, then amplify with vigorous shaking at 220-250 rpm at 37°C for 4.5-5 hours;

[0042] 4. Collect the culture in a sterilized centrifuge cup, pre-cool at 4°C, and centrifuge at 10,000rpm for 10min;

[0043] 5. Transfer the supernatant phage solution obtained after centrifugation to a new centrifuge cup, add 1 / 5 volume of PEG / NaCl (about 40mL), mix well and place it at 4°C overnight (about 16-18h) to make the phage Precipitation is ...

Embodiment 3

[0050] Phage titer determination:

[0051] 1. Preparation of the lower IPTG / X-gal / LB plate: Heat and melt 100mL solid LB medium and cool it to about 55°C, add 100μL IPTG / X-gal stock solution evenly, mix quickly and pour the sterile plate (Φ = 0.9 cm). After cooling, wrap with plastic wrap and pre-warm at 37°C for later use;

[0052] 2. Preparation of the upper layer of LB semi-solid agar: Heat and melt the semi-solid LB medium and cool it to 55°C, divide each 3mL into sterile EP tubes, keep it in a water bath at 45°C for later use;

[0053] 3. Transfer the ER2738 cultured overnight in Step 3 of 1.2.1 to LB liquid medium at a ratio of 1:100 (2-3 mL of culture can be prepared in total), and shake vigorously at 37°C for about 2 hours to make it reach the desired level. number of growing seasons;

[0054] 4. Dispense the ER2738 in the logarithmic growth phase into 1.5mL sterilized Eppendorf (EP) at 200μL / cartridge for later use;

[0055] 5. Prepare serial dilutions of phage so...

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Abstract

The invention discloses a PD-1 targeted blocking peptide and an application thereof, and belongs to the field of antitumor immunity treatment. A phage display technique is adopted for obtaining the blocking peptide for specially targeting PD-1, and the sequence of the blocking peptide is shown as SEQID NO.1. Phages are used for showing a 12-peptide library, and through four-round biological elutriation, phage monoclonal antibodies which have specificity affinity interaction with PD-1 protein are obtained and can target tumor cells naturally expressing PD-1; through sequencing, a polypeptide sequence shown on the surfaces of the phages can be obtained; and is chemically synthesized so that the polypeptide sequence is proved to be capable of adjusting human PBMC to secrete cell factors IFN-gamma and IL-2; and the PD-1 blocking peptide is entrapped, so that nanoparticles are prepared. In-vivo drug effect experiment verifies that the PD-1 targeted blocking peptide has notable antitumor effects. The PD-1 targeted blocking peptide can realize specific binding with the PD-1, combination of PD1 and PD-L1 can be blocked, and the functions of T cells can be restored. The targeted blocking peptide can be widely applied to the fields of tumor targeting, immunomodulation and tumor resistance.

Description

technical field [0001] The invention relates to a targeted blocking peptide capable of specifically binding to PD-1, and belongs to the field of biotechnology. It specifically relates to the biological panning method of the blocking peptide, the targeting effect at the cell level and the immune stimulating effect and its application in anti-tumor research. Background technique [0002] Phage display technology was created by George P.Smith in 1985. By engineering phages, foreign polypeptides or proteins are fused and expressed with certain capsid proteins of phages, while ensuring their ability to infect and reproduce. , so that the exogenous amino acid sequence is displayed at the end of the capsid protein. The random dodecapeptide phage display library is a combinatorial library that fuses random dodecapeptides to the minor capsid protein (pIII) of M13 phage. The displayed dodecapeptide is expressed at the N-terminus of pill. Through at least three rounds of biological ...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K19/00A61K38/10A61P35/00A61P37/02
CPCC07K7/08A61P35/00A61P37/02C07K2319/00A61K38/00Y02P20/55
Inventor 邱郑王旻陶慧敏
Owner CHINA PHARM UNIV
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