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Trastuzumab-modified toxin protein-coated PEG immune liposome and preparation and application thereof

A technology of immunoliposome and toxin protein, which is applied in the field of PEGylated immunoliposome encapsulating toxin protein, can solve the problems of large molecular weight, loss of activity, complex spatial structure, etc., to reduce sensitivity, reduce exposure, and reduce non-specific Heterotoxic effects

Inactive Publication Date: 2009-09-23
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, protein drugs have the characteristics of large molecular weight and complex spatial structure. During the drug delivery process, they are easily affected by the complex physiological environment (especially the action of a large number of enzymes), which will damage the structure of the protein and lead to its activity. lost

Method used

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  • Trastuzumab-modified toxin protein-coated PEG immune liposome and preparation and application thereof
  • Trastuzumab-modified toxin protein-coated PEG immune liposome and preparation and application thereof
  • Trastuzumab-modified toxin protein-coated PEG immune liposome and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Separation and purification of anti-tumor toxin protein PE38KDEL

[0037] PE38 is a type of pseudomonas extoxin (PE) among bacterial toxins, with a molecular weight of 38KD. PE38KDEL is a preferred PE38 mutant, which has the advantage of low non-specific toxicity (provided by the Cancer Institute of Second Military Medical University) .

[0038] Referring to the literature Song S, Xue J, Fan K, et al. Preparation and characterization of fusionprotein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli. Protein Expr Purif 2005; 44: 52-57. Method, first construct the pET of PE38KDEL (Novagen) The expression vector was then transferred into the engineering bacteria BL21(DE3) (Novagen), and the PE38KDEL toxin protein was induced and expressed by the inducer IPTG (isopropyl-β-D-thiogalactoside, isopropyl-β-D-thiogalactoside) . Then, PE38KDEL soluble in bacteria was obtained by nickel column affinity chromatography. Finally, the in vitro protein tran...

Embodiment 2

[0039] Example 2: Preparation of PEGylated liposomes encapsulating anti-tumor toxin protein PE38KDEL

[0040] The concentration of the aqueous solution of the isolated and purified toxin protein PE38KDEL was precisely measured by the MicroBCA method (Pierce Company), and the concentration range was preferably 2-10 mg / ml. EPC, CHOL, mPEG 2000 -DSPE (2: 1: 0.08, molar ratio) was dissolved in 2-3 ml of chloroform solution, the solution was added into a pear-shaped flask, filled with nitrogen three times, and then the organic solvent was completely evaporated with a rotary evaporator under vacuum conditions. The prepared lipid film was fully hydrated with 2mg / ml PE38KDEL solution to obtain a liposome suspension, and then passed through 800nm, 400nm, and 200nm polycarbonate membranes with a film extruder, and then washed with PBS (pH 7.4) is the eluent through the Sepharose CL-4B column to remove unwrapped PE38KDEL, and the collected sample is concentrated in a 1000,000Da ultrafil...

Embodiment 3

[0042] Example 3: Modification of Antibodies and Linkage of Modified Antibodies to Liposomes

[0043] (1) Preparation of MPB-Ab (maleimidophenylbutyrate-HER2)

[0044] Ab (antibody Trastuzumab, trade name Herceptin, purchased from Genentech) was dissolved in HEPES buffer (25mM HEPES, 140mM NaCl, pH 7.4) to prepare a 10mg / ml Ab solution. Dissolve 25 mM of SMPB (N-succinimidyl-4-(p-maleimidophenyl)-butyrate, N-succinimidyl-4-p-maleimidophenylbutyrate) in DMF (dimethylformamide solution), and then slowly added to the above Ab solution (the molar ratio of SMPB to Ab was 20:1), and reacted at room temperature for 30 minutes. Then use HEPES and MES (25mM HEPES, 25mM MES, 140mM NaCl, pH 6.7) as the eluent to remove the unbound SMPB through the Sephadex G50 column. After concentration at ℃, a 10 mg / ml MPB-Ab solution was obtained, and MPB-BSA (bovine serum albumin) was prepared in the same way as a control.

[0045] (2) Binding of antibodies

[0046] PEGylated immunoliposomes were...

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Abstract

The invention relates to the technical field of medicine. The traditional tumor treatment methods, such as radiation treatment, chemical treatment, biotic factor treatment, and the like are lack of targeting, and the immunotoxin treatment has the problem of sensibility of the nonspecific toxicity and the immunogenicity of toxic proteins, etc. The humanized McAb Trastuzumab-modified toxin protein-coated PEG immune liposome has the structure: an antibody is connected to the surface of lipidosome and can be combined with HER 2 antigen on the surface of a tumor cell so as to generate endocytosis; and the toxin proteins are coated in the lipidosome, and the sensibility of the nonspecific toxicity and the immunogenicity of the toxic proteins can be reduced. The invention also provides the preparation and the application of the lipidosome. Proved by the result of the experiment of killing the tumor cells in vitro, the biological targeting is favorable, and the function of killing the targeted tumor cells in vitro is outstanding; and the lipidosome has low toxic effect on normal cells.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a PEGylated immunoliposome encapsulated with toxin protein modified by humanized monoclonal antibody Trastuzumab, and its preparation and application. Background technique [0002] Traditional tumor treatment methods include: radiation therapy, chemotherapy, biological factor therapy and so on. However, none of these therapeutic methods specifically target tumor cells, that is, they lack targeting. While the drug kills tumor cells, it also produces extensive toxicity to normal tissue cells. As a result, the availability of the drug is limited, the side effects are large, and the dosage is limited. Therefore, there is a need for an effective targeted drug delivery system to allow the drug to reach the tumor area. This system can not only increase the concentration of drugs in the tumor area, kill tumor cells more effectively, but also reduce the damage to normal cells and tiss...

Claims

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Application Information

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IPC IPC(8): A61K9/127A61K47/48A61K38/16A61P35/00A61K47/68
Inventor 钟延强钟威郭亚军高洁张翮鲁莹李博华方晨周闺臣孙霁邹豪孙治国王江峰王明娟刘青锋陈婷宣吉明
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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