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Protein drug combined with immunosuppressive molecule PD-L1

An immunosuppressive molecule, the technology of PD-L1, applied in the field of medical science, can solve the problems that the development of covalent protein drugs has not made effective progress, and achieve the effect of improving binding ability and inhibiting cancer cells

Active Publication Date: 2020-12-04
GUANGDONG SHENGSAI BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the development of covalent protein drugs has not made effective progress

Method used

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  • Protein drug combined with immunosuppressive molecule PD-L1
  • Protein drug combined with immunosuppressive molecule PD-L1
  • Protein drug combined with immunosuppressive molecule PD-L1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Covalent binding of PD1FSY to PD-L1 protein

[0027]Preparation of PD1 (WT), PD1 (Q75FSY), PD1 (D77FSY), PD1 (A129FSY) protein, designed to express PD1 IgV domain (residues 32-160, cysteine ​​93 replaced by serine to improve protein stability sex), the structure is PD1 (WT); the TAG codons introduced at the corresponding positions of amino acids 77, 129 and 75 are PD1 (D77FSY), PD1 (A129FSY), PD1 (Q75FSY), and the optimized The DNA sequence of the His tag was cloned into the pET-26b plasmid. For PDL1 (WT), the DNA sequence from 19 to 134 residues of PDL1 protein was cloned into the pET-26b plasmid, and in PDL1 (H69A), histidine 69 was replaced with alanine, and the optimized sequence was also cloned into pET- 26b plasmid. The above plasmids were introduced into Escherichia coli BL21(DE3) by electroporation. For PD1(WT), PD-L1(WT) and PD-L1(H69A), the plasmid-transfected bacteria used 2×YT medium (add 50ug / ml kanamycin, 1mM IPTG) cultured at 37°C, for expre...

Embodiment 2

[0029] Example 2 Covalent binding of PD1FSY to tumor cell PD-L1 molecules

[0030] will be 5×10 5 H460 and U87 tumor cells were respectively plated in 6-well plates. After 6 hours, PD1(WT) and PD1(A129FSY) proteins were incubated with the above two kinds of cells at 37°C for 12 hours. After reaching the time point, they were digested with trypsin and collected. Cells, and use 100ul RIPA lysate to lyse the cells, extract the total protein, add 5× protein loading buffer, denature at high temperature (100°C) for 10 minutes, separate the sample by 15% SDS-PAGE gel, and transfer the protein to the NC membrane On (90V, 1.5 hours), use 5% skimmed milk to block for one hour, use anti-PD-L1 and β-Actin antibodies to incubate overnight at 4°C, wash the membrane three times with TBST and use anti-mouse or rabbit IgG with HRP The secondary antibody was incubated for 1 hour, the membrane was washed three times, and the bands were exposed. see results image 3 .

Embodiment 3

[0031] Example 3 Covalent binding of PD1FSY to tumor tissue PD-L1 molecules

[0032] Take 2×10 6 Tumor cells H460 and U87 were transplanted into severe immunodeficiency mice (NSG) by subcutaneous injection, and 10 days later, the drugs PD1 (WT) and PD1 (A129FSY) were subcutaneously injected into the mice through the tail vein and the tumor paratumours, respectively. Hours later, the mice were treated with CO 2 After being anesthetized and sacrificed by cervical dislocation, the tumor tissue was collected, and the total tumor protein was extracted with 200ul of RIPA lysate. The cross-linking detection of PD-L1 was the same as the western blot detection method in Example (2). see results Figure 4 .

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Abstract

The invention provides a non-natural amino acid modified protein drug combined with immunosuppressive molecule PD-L1. The protein drug is integrated into a covalent protein drug through non-natural amino acid with potential biological activity, and is combined with a target protein through an accessible reaction, and the non-natural amino acid can be covalently combined with a target protein aminoacid residue. The non-natural amino acid modified PD-1 enhances the binding capacity with PD-L1, has the effect of inhibiting cancer cells, and has the effect of treating tumors.

Description

technical field [0001] The invention belongs to the field of medical science and technology, and relates to a protein drug combined with immunosuppressive molecule PD-L1. Background technique [0002] With the development of biotechnology, the treatment methods for malignant tumors are constantly improving. In addition to traditional treatment methods such as surgery, major breakthroughs have been made in chemotherapy and radiotherapy; in terms of drug development, the update and iteration speed of new drugs is constantly accelerating. In the field of tumor immunotherapy, drugs related to immune activation and inhibition pathways have initially shown their application prospects, such as immune checkpoint inhibitors, PD1-PD-L1, CTLA4, TIM3, LAG3 and other inhibitors. In the immune microenvironment of solid tumors, tumor cells or related suppressive immune cells induced by tumors resist immunotherapy through the braking effect of the immune system, which greatly reduces the ef...

Claims

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Application Information

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IPC IPC(8): C07K14/705A61K38/17A61P35/00
CPCC07K14/70521A61P35/00A61K38/00
Inventor 徐洋王磊陈渠
Owner GUANGDONG SHENGSAI BIOTECH CO LTD
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