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PD-L1 targeting polypeptide and application thereof

A PD-L1, peptide targeting technology, applied in applications, medical preparations containing active ingredients, specific peptides, etc., can solve problems such as poor efficacy, improve tumor immunity, avoid interactions, and activate T cells effect of immune response

Active Publication Date: 2018-11-20
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

[0005] In view of the above-mentioned defects of the prior art, the purpose of the present invention is to address the deficiencies of the prior art and the demand for treating tumors, and solve the existing immune response based on the combination of PD-L1 antibody and PD-L1 to activate T cells, so as to achieve There are problems such as poor curative effect in the method of improving anti-tumor immune activity. The present invention provides a polypeptide capable of reducing the expression of PD-L1 protein in tumor cells, an isolated nucleic acid, a nucleic acid construct, a recombinant expression vector, and an anti-tumor drug. and its application

Method used

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  • PD-L1 targeting polypeptide and application thereof
  • PD-L1 targeting polypeptide and application thereof
  • PD-L1 targeting polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Confirmation of the PD-L1-interacting amino acid sequence of the PD-L1-targeting polypeptide and the key amino acid sequence targeting lysosome degradation

[0033] 1. Confirm that HIP1R promotes the degradation of PD-L1 through lysosomal pathway:

[0034] HIP1R eukaryotic expression vector was transfected in human colorectal cancer cells HCT116, and the expression level of PD-L1 protein was detected by immunoblotting method with PD-L1 specific antibody, and it was found that HIP1R significantly reduced the expression of PD-L1 protein The effect of HIP1R was effectively inhibited by blocking the lysosomal degradation pathway with different doses of chloroquine ( image 3 A). The cells were treated with chloroquine, a specific inhibitor of the lysosomal degradation pathway (chloroquine concentrations were 5, 10, 20, 40 μM), and it was found that HIP1R could be inhibited in a dose-dependent manner ( image 3 A). like image 3 As shown in B, the overexpressi...

Embodiment 2

[0040] Example 2. Construction of PD-L1-targeting polypeptide and its functional verification

[0041] 1. Construction of peptides targeting PD-L1:

[0042] According to the previous experiments, the (784-807) sequence of HIP1R binds to PD-L1, while the HIP1R (1059-1068) sequence targets lysosomes, which can promote the degradation of PD-L1 protein in the lysosomal pathway. Utilizing the above original findings, a polypeptide that binds to PD-L1 and targets lysosomal degradation was designed, namely HIP1R(784-807)-(1059-1068), which was named PD-LYSO for the convenience of the following description. Its composition structure and specific sequence are as follows Image 6 shown. At the same time, as a control, PD-LYSO (mut) was constructed, which lacked part of the N-terminal amino acid sequence of PD-LYSO, and PD-LYSO (plus), which added part of the amino acid sequence to the N-terminal of PD-LYSO. Image 6 It is a gel electrophoresis image verifying the actual effect of the...

Embodiment 3

[0046] Example 3. Functional verification of PD-L1-targeting polypeptides in reducing PD-L1 expression in cells

[0047] 1. The verification that peptides targeting PD-L1 reduce cancer cell binding to PD-1

[0048] Peptides targeting PD-L1 can reduce the binding ability of PD-L1 on the surface of tumor cells. After the tumor cells were treated with peptides for 24 hours, they were incubated with the PD-1 protein fused to the Fc region of the antibody to bind, and then the amount of bound PD-1 was detected by anti-PD-1 antibody flow cytometry. The statistical results are as follows: Figure 8 shown. Tumor cells bind to PD-1 on the surface of T cells through PD-L1 expressed on the cell surface, thereby inhibiting the killing effect of T cells. Therefore, the ability of PD-L1 targeting polypeptide (PD-LYSO) to bind PD-1 to cancer cells was first verified. After the tumor cells were treated with the polypeptide for 24 hours, they were incubated with the PD-1 protein fused to th...

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Abstract

The invention provides PD-L1 protein targeting polypeptide, isolated nucleic acid, a nucleic acid construct, a recombinant expression vector and an antitumor medicine, and further provides applicationof the PD-L1 protein targeting polypeptide to preparation of the antitumor medicine. The polypeptide has the function of regulating PD-L1 protein, and can reduce the expression quantity of the PD-L1protein in cells, reduce the PD-1 protein bonding ability of tumor cells, increase the tumor cell killing ability of T cells and inhibit the in-vivo growth of the tumor cells. Different from the current commonly-used antibody blocking method, a technical scheme provided by the invention is beneficial to activation of T cell immune response and improvement on tumor immunity, is a new breakthrough as a tumor immunotherapeutic method, and has a wide meaning in biomedical and pharmaceutical research in allusion to PD-L1 regulation and control.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a polypeptide for targeting PD-L1 protein, an isolated nucleic acid, a nucleic acid construct, a recombinant expression vector, an antitumor drug and applications thereof. Background technique [0002] Malignant tumors are a type of disease with the highest morbidity and mortality in my country. In recent years, the development of clinical diagnosis, surgery, radiotherapy and chemotherapy has enabled some patients with malignant tumors to receive early detection and effective treatment. However, searching for new treatment methods and drugs has long been a hot spot in the field of cancer research all over the world. 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, establish immune memory, and prevent tumor recurrence, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/71C12N15/12A61K38/17A61P35/00
CPCA61K38/00A61P35/00C07K14/71
Inventor 许杰王焕彬姚晗李楚舒章瑶石虎兵房静远陈萦晅
Owner RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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