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Polypeptide for inhibiting PD-L1 palmitoylation modification and expression and application of polypeptide

A technology of PD-L1 and palmitoylation, which is applied to polypeptides containing positioning/targeting motifs, chemical instruments and methods, peptides, etc., can solve the problems of reduced curative effect and poor effect, and achieve good inhibition principles and effects , the effect of reducing the content of PD-L1

Active Publication Date: 2020-01-21
RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the current immune checkpoint blockade therapy based on anti-PD-L1 antibody still has great challenges and shortcomings. For example, the effect of anti-PD-L1 antibody therapy on some tumor patients is not good, and its efficacy may vary Decreased with prolongation of medication time

Method used

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  • Polypeptide for inhibiting PD-L1 palmitoylation modification and expression and application of polypeptide
  • Polypeptide for inhibiting PD-L1 palmitoylation modification and expression and application of polypeptide
  • Polypeptide for inhibiting PD-L1 palmitoylation modification and expression and application of polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 identifies the site and basic sequence of palmitoylation of endogenous PD-L1 expressed by tumor cells, and proves that palmitoylation inhibits the degradation of PD-L1 by lysosomes.

[0040] (1) Identify the site and basic sequence of palmitoylation of PD-L1:

[0041] The endogenous PD-L1 expressed in HCT116 human colorectal cancer cells was purified by immunoprecipitation, gel electrophoresis was performed, and the Coomassie brilliant blue stained band corresponding to the molecular weight of PD-L1 was cut, digested with trypsin, and analyzed by liquid chromatography. The obtained peptides were analyzed by mass spectrometry and compared with the FindMod database to identify post-translational modifications of PD-L1. Such as image 3 As shown, the cysteine ​​C272 at position 272 in the endogenous PD-L1 expressed by HCT116 cells undergoes palmitoylation.

[0042] (2) Proving that palmitoylation inhibits the lysosomal degradation of PD-L1.

[0043] In order t...

Embodiment 2

[0044] Example 2 Design of PD-L1 palmitoylation inhibitory polypeptide S1 (PD-palm), its localization and expression in cells, and its effect on PD-L1 expression.

[0045] (1) Using the principle of competitive inhibition to design PD-palm, a polypeptide inhibiting palmitoylation of PD-L1.

[0046] Given that the palmitoylation site of PD-L1 is C272, the short peptide motif modified by palmitoylation is usually determined by the common characteristics of 14 amino acids near the modification site. Therefore, the PD-L1 (265-279) short peptide was designed as a "bait" that can undergo palmitoylation modification, and the principle of competitive inhibition was used to reduce the palmitoylation modification of endogenous PD-L1.

[0047] Such as figure 1 and figure 2 As shown, through the EcoRI and Xhol cloning sites, the nucleic acid sequence (SEQ ID NO: 2) encoding the polypeptide S1 (PD-palm) sequence (SEQ ID NO: 1) in the nucleic acid construct was inserted into the eukaryot...

Embodiment 3

[0051] Example 3 Using cell-penetrating peptides to transport PD-palm polypeptides synthesized in vitro into cancer cells to promote the degradation of PD-L1

[0052] The cell-penetrating peptide CPP can effectively carry polypeptides synthesized in vitro into cells, so if Figure 9 As shown, the fusion polypeptide CPP-S1 (PD-palm) of CPPtat and PD-palm and CPP-S2 as a control were constructed, wherein CPPtat is HIV-1 virus TAT ​​protein transduction peptide. Synthesized by the standard Fmoc solid-phase synthesis method, after purification by high-performance liquid chromatography, CPP-S1 (PD-palm) was identified by mass spectrometry, and the identification results were as follows Figure 10 shown.

[0053] CPP-S1 (PD-palm) and CPP-S2 were added to HCT116 human colorectal cancer cell culture medium at different final concentrations (0, 1.2, 2.5, 5, 10 μM), incubated for 24 hours, and analyzed by immunoblotting Methods To detect the expression of PD-L1. Such as Figure 11 A...

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Abstract

The invention discloses a polypeptide for inhibiting PD-L1 palmitoylation modification and expression and application of the polypeptide and relates to the technical field of biological pharmacy. Thepolypeptide is a fusion polypeptide of a cell-penetrating peptide and a PD-L1 palmitoylation-inhibiting polypeptide; the cell-penetrating peptide is a TAT protein transduction peptide; and the PD-L1 palmitoylation-inhibiting polypeptide is a PD-L1 protein-targeting targeting peptide. The polypeptide disclosed by the invention implements competitive inhibition on endogenous PD-L1 palmitoylation modification of tumor cells, induces PD-L1 proteins to be degraded and cleared by a lysosome and reduces the contents of PD-L1 on the surfaces of the tumor cells and in the cells at the same time. Compared with an anti-PD-L1 antibody, the polypeptide disclosed by the invention fundamentally lowers the content of the Pd-L1 in the tumor cells and is more beneficial to overcoming the immune escape function obtained by the tumor cells through expression of the Pd-L1.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a polypeptide that inhibits PD-L1 palmitoylation modification and expression and a preparation method thereof. Background technique [0002] Malignant tumors are a class of diseases with the highest morbidity and mortality in my country. Although the development of clinical diagnosis, surgery, radiotherapy and chemotherapy in recent years has enabled some patients with malignant tumors to receive early detection and effective treatment, the search for new drugs and new treatment methods is still a long-term focus in the field of cancer research worldwide. Different from traditional treatment methods, tumor immunotherapy can activate or induce the body to establish a specific immune response to tumor antigens, eliminate primary or metastatic tumor cells, and establish immune memory to prevent tumor recurrence, drug resistance, and metastasis. Programmed death ligand 1 ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62
CPCC07K7/08C07K2319/10Y02P20/55
Inventor 许杰姚晗王焕彬李楚舒
Owner RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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