Mutants of highly active tumor necrosis factor-related apoptosis-inducing ligands

A tumor necrosis factor and apoptosis-inducing ligand technology, which can be used in anti-tumor drugs, receptors/cell surface antigens/cell surface determinants, pharmaceutical formulations, etc., and can solve problems such as low binding force

Active Publication Date: 2019-03-05
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The third type is the soluble receptor OPG (osteoprotegrin). Although it can bind to TRAIL and inhibit its activity, its binding ability to TRAIL is very low under normal physiological conditions (Curr Opin Pharmacol 2008,8:433-439)
[0005] The combination of TRAIL and the death receptors DR4 and DR5 on the surface of tumor cell membrane can induce tumor cell apoptosis, but at the same time TRAIL can also combine with decoy receptors DcR1, DcR2 and OPG, but this combination cannot induce tumor cell apoptosis

Method used

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  • Mutants of highly active tumor necrosis factor-related apoptosis-inducing ligands
  • Mutants of highly active tumor necrosis factor-related apoptosis-inducing ligands
  • Mutants of highly active tumor necrosis factor-related apoptosis-inducing ligands

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Design of DR5 high-affinity TRAIL mutants

[0031] Construction of the TRAIL-R complex model: the TRAIL-DR5 complex (PDB ID: 1D4V) was used as a template, which is a monomeric complex of TRAIL and DR5 extracellular domain, with a resolution of Wherein DR5 includes amino acids 69-184, and the structure of the TRAIL-DR5 trimer complex was generated using the PDBePISA server (http: / / pqs.ebi.ac.uk) of the EBI website. The structure of the TARIL-DR5 trimer complex obtained in this way must have some unreasonable energy collisions, so the MOE (Molecular Operating Environment, Chemical Computing Group, Montreal) software was used to optimize the energy of the pair: first, the system was added to the system with Protonate 3D Hydrogen, charge, and then under the OPLS-AA force field, set the Gradient to 0.05, and perform energy minimization. The optimized structure is used as the initial structure for subsequent FoldX (http: / / foldxsuite.crg.eu / , Center for Genomic Reg...

Embodiment 2

[0040] Example 2 Construction of TRAIL (114-281) and its mutant protein engineering bacteria

[0041] Construction of TRAIL(114-281) expression strain. Design and synthesize the following primers: P1(5-CC GAATTC ATGGTGAGAGAAAGAGGTCCTCAGAGAG-3, SEQ ID NO: 6, the underline is the introduced EcoR I restriction site) and reverse primer P2 (5-AC AAGCTT AGCCAACTAAAAAGGCCCCGAAAAAACTG-3, SEQ ID NO: 7, the underline is the introduced Hind III restriction site). Human placenta cDNA was used as template, P1 and P2 were used as upstream and downstream primers, respectively, and high-fidelity TaqDAN polymerase PCR was used to amplify the TRAIL (114-281) coding gene. PCR conditions were: denaturation at 94°C for 5 minutes; 30 cycles at 94°C for 30s, 50°C for 50s, and 72°C for 90s; extension at 72°C for 10 minutes. After purification, the PCR product was cloned into the pMD19-T vector (product of Dalian Biotech Co., Ltd.) and sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing...

Embodiment 3

[0043] Example 3 Expression of TRAIL(114-281) and its mutant protein

[0044] A single colony of the recombinant engineered bacteria was placed in 50 ml LB liquid medium (containing 100 μg / ml ampicillin) at 37° C., 220 rpm, and cultured for 12 hours. Inoculate 200ml fermentation medium (1% tryptone, 0.5% yeast powder, 4% sodium glutamate, 1% malt powder, 0.67% KH 2 PO 4 , 0.75% Na 2 HPO 4 . 12H 2 O, 0.1 mM ZnSO 4 , 100 μg / ml ampicillin, pH 6.5), 37°C, 220rpm, cultured for 24h.

[0045] The fermentation broth was centrifuged (6000rpm, 10min) to collect the bacteria sludge (wet bacteria weight, 6g / l), and resuspended in the bacteriostasis buffer (20mM Tris-HCl, 0.05mM ZnSO 4, 0.25mM DTT, 0.1% Triton, pH 7.6), the cells were disrupted with a homogenizer (AH100B, ATS Engineering Inc., Canada). The broken liquid was centrifuged (13000rpm, 30min) to collect the supernatant. 10% SDS-PAGE electrophoresis analysis of the total protein of the bacteria, the supernatant of the br...

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Abstract

The invention belongs to the field of biological pharmacy and particularly relates to a mutant of a TRAIL (TNF (tumor necrosis factor)-related apoptosis-inducing ligand) and a pharmaceutical application of the mutant. Due to the fact that a wild type TRAIL can be simultaneously combined with tumor cell membrane surface DR (death receptor)4 and DR5 as well as a DcR (decoy receptor)1 and a DcR2 and can be combined with a soluble receptor OPG (osteoprotegrin) by the aid of very low affinity, but tumor cell apoptosis can be induced only through combination of the wild type TRAIL with the DR4 and the DR5; in order to enhance the anti-tumor activity of the TRAIL, the TRAIL mutant having high affinity with the DR5 is designed and constructed, and the TRAIL mutant is characterized by containing E263G mutation, that is, the 263th Glu is mutated into Gly. SEC-HPLC (size exclusion chromatography-high performance liquid chromatography) analysis indicates that the mutant exists in a form of a tripolymer with activity after the mutant is subjected to soluble expression and purification in E.coli. The affinity of the mutant with the DR5 is remarkably improved, and the anti-tumor activity of the mutant is further remarkably improved.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mutant, which has higher affinity to the death receptor DR5 on the surface of tumor cells than wild-type TRAIL , while exhibiting higher antitumor activity than wild-type TRAIL. Background technique [0002] Tumor necrosis factor-related apoptosis-inducing ligand (TNF-related apoptosis-inducing ligand, TRAIL) also known as apoptin 2 ligand (Apoptosis Ligand, APO-2L) is a member of the tumor necrosis factor (TNF) family, it and TNF Like other members of the superfamily, they belong to type II transmembrane proteins. Since TRAIL can specifically induce tumor cell apoptosis and has little cytotoxicity to normal cells, it has broad application prospects in tumor immunotherapy (Cancer Lett 2013, 332:156-162). [0003] The human TRAIL gene encodes a total of 281 amino acids, its relative molecular weight is 32.5KD, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/705A61K38/17A61P35/00
CPCA61K38/00C07K14/70575
Inventor 谭树华付剑黄传龙古丽丽孙玥盈朱娟娟
Owner CHINA PHARM UNIV
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