Tumor targeting tumor necrosis factor related apoptosis ligand variant and application thereof

A tumor necrosis factor, tumor-targeting technology, used in anti-tumor drugs, for targeting specific cell fusion, receptors/cell surface antigens/cell surface determinants, etc., can solve side effects, unpredictable immune results, etc. To achieve the effect of prolonging half-life, good pharmacokinetic effect, and good tumor tissue targeting

Inactive Publication Date: 2013-01-09
JIANGSU TARGET BIOMEDICINE RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, repeated and systemic use of high-dose exogenous TNF-related apoptosis ligand protein in clinical practice may lead to unpredictable immune results
These reports have made scientists very concerned about the potential side effects of tumor necrosis factor-related apoptosis ligands when used repeatedly and persistently in the clinic

Method used

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  • Tumor targeting tumor necrosis factor related apoptosis ligand variant and application thereof
  • Tumor targeting tumor necrosis factor related apoptosis ligand variant and application thereof
  • Tumor targeting tumor necrosis factor related apoptosis ligand variant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Preparation of tumor-targeting tumor necrosis factor-related apoptosis ligand variants

[0045] Using computer-aided structural simulation and molecular design, on the SGI computer workstation, using MSI company's molecular design software (InsightII, Discover and other modules), based on the crystal structure of tumor necrosis factor-related apoptosis ligands, the Add CD13 ligand short peptide to the N-terminal of the apoptosis ligand, carry out molecular modeling and molecular design, and determine the amino acid length of the connecting peptide.

[0046] On the basis of the above molecular design, we first chose the design scheme of linking peptide with 5 glycines, because computer simulation showed that the linking peptide of this scheme is shorter, and it has a great impact on the structure of tumor necrosis factor-related apoptosis ligand protein, CD13 ligand, etc. The combination of body and CD13 may have a greater impact. Most of the connecting peptides of othe...

Embodiment 2

[0058] Binding assay of tumor necrosis factor-related apoptosis ligand and its tumor-targeting variants to endothelial cells

[0059] Tumor necrosis factor-related apoptosis ligands and their tumor-targeting variants NGR-L-TRAIL and RGD-L-TRAIL variants were labeled with fluorescein (Sigma) respectively, and the labeled proteins were passed through Sephadex-G25 molecular sieve Remove free fluorescein. Human foreskin microvascular endothelial cells were digested with trypsin, washed twice with ice-cold phosphate buffer containing 2% fetal bovine serum, then resuspended, added 1 microgram of labeled protein, and incubated at 4°C for 1 hour. Fluorescein-labeled bovine serum albumin was used as a control in the experiment. After the stained cells were washed three times, the binding capacity was analyzed by flow cytometry (Becton Dickinson Company).

[0060] We further evaluated the fluorescein-labeled tumor necrosis factor-related apoptosis ligand and its tumor-targeting varian...

Embodiment 3

[0062] Expression Analysis of CD13 and Integrin αVβ3, αVβ5

[0063] The expression abundance of CD13 or integrin on the cell surface was detected by flow cytometry analysis by indirect labeling method. The digested cells were washed twice with ice-cold phosphate buffer containing 2% fetal bovine serum, resuspended and coated with anti-human CD 13 monoclonal antibody (eBioscience) or anti-human αVβ3 integrin antibody MAB23C6 (eBioscience) on ice. , company) or anti-human αVβ5 integrin antibody MAB 1961 (Chemicon International Company) for 1 hour, and the negative control was purified isotype mouse immunoglobulin G (eBioscience Company). After the primary antibody-coated cells were washed twice, green fluorescein-conjugated goat anti-mouse immunoglobulin G1 (γ) (Caltag Laboratories) secondary antibody was added to label in the dark for 30 minutes. Then wash three times, wash and fix with phosphate buffer solution containing 4% formalin, and finally detect and analyze the abunda...

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Abstract

The invention belongs to the technical field of biological genetic engineering and particularly discloses design, a preparation method and pharmaceutical application of a tumor targeting tumor necrosis factor related apoptosis ligand variant. The tumor targeting tumor necrosis factor related apoptosis ligand variant is fusion protein composed by ligand, connecting peptide and tumor necrosis factor related apoptosis ligand of CD13 and is produced through cloning and constructing of coding genes of the variant, soluble recombination expression and simple separation and purification in a genetic engineering method. The variant generated by the preparation method of the tumor targeting tumor necrosis factor related apoptosis ligand variant has good tumor tissue targeting property, remarkably enhances antitumous effect, can reduce protein administration dosage required for obtaining therapeutic effect, improves bioavailability, lowers treatment cost, and eliminates potential toxic and side effect of the tumor necrosis factor related apoptosis ligand. Simultaneously, the manufacturing method of the tumor targeting tumor necrosis factor related apoptosis ligand variant provides an expression method for producing soluble high-polymer form content and a simple separation and purification process.

Description

technical field [0001] The invention belongs to the field of genetic engineering biotechnology, and relates to the preparation and pharmaceutical application of a tumor-targeting tumor necrosis factor-related apoptosis ligand variant. Background technique [0002] Tumor necrosis factor-related apoptosis ligand (TRAIL) is a member of the tumor necrosis factor superfamily. Similar to other members of the tumor necrosis factor superfamily, soluble tumor necrosis factor-related ligands bind to trimerized receptor molecules on the surface of target cells in the form of trimers to exert biological functions. Tumor necrosis factor-related apoptosis ligand induces apoptosis through the interaction with death receptor 4 (DR4) and death receptor 5 (DR5) on tumor cells to transmit death signals. Although other members of the tumor necrosis factor superfamily have limited application due to systemic toxic side effects, tumor necrosis factor-related apoptosis ligand is a relatively safe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/22C07K1/18C12N15/62C12N15/70A61K38/17A61K48/00A61K47/48A61P35/00
CPCA61K47/48C07K2319/00A61K48/00C07K19/00C12N15/62A61K38/17C07K14/70575C12N9/485C07K5/0819C07K1/22C12Y304/11002C12N15/70C07K1/18C07K2319/33C07K2319/74A61K38/00A61P35/00A61P43/00
Inventor 华子春曹林唐波
Owner JIANGSU TARGET BIOMEDICINE RES INST
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