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Preparation of a new generation of multifunctional antibody nanoclusters and their synergistic therapeutic applications

A nano-cluster and antibody technology, applied in the field of medicine, can solve the problems of tumor cell targeting and binding, insufficient tumor enrichment, uncontrollable drug release, etc., to achieve strong tumor killing effect and enhance anti-tumor effect Effect

Active Publication Date: 2017-09-29
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

However, the development of the traditional nano-drug delivery system is still not perfect, and is limited by many factors in vivo and in vitro, resulting in the bottleneck problems of current nano-drug treatment: 1. Nano-carriers have poor stability in vivo and short circulation time; 2. Due to the targeting and stability 3. Poor targeting and binding of tumor cells leading to less endocytosis; 4. Uncontrollable release of drugs from specific intracellular sites
[0006] The novel antibody nano-cluster designed by the present invention has no similar research reports at home and abroad

Method used

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  • Preparation of a new generation of multifunctional antibody nanoclusters and their synergistic therapeutic applications
  • Preparation of a new generation of multifunctional antibody nanoclusters and their synergistic therapeutic applications
  • Preparation of a new generation of multifunctional antibody nanoclusters and their synergistic therapeutic applications

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1: Preparation method of anti-CD20 antibody nanoclusters

[0050] (1) Prepare PEI (polyethyleneimine, molecular weight 25kDa) and MPEGS (MAL-PEG-SCM, maleimide-polyethylene glycol-succinimide acetate) solution of 4mg / ml respectively, will PEI and MPEGS were mixed at a mass ratio of 1:20. After fully reacting at room temperature for 4 hours, they were dialyzed with a 3500MCW dialysis membrane for 4-6 hours to remove unreacted free MPEGS.

[0051] (2) Thiolated antibody: Take 1mg of Rituximab and 11B8 antibody stock solutions and dilute them to 2mg / ml respectively. According to 2-IT / mAbs=0.15:1, add 5mg / ml 2-IT (2-iminothiolane) to the antibody solution respectively, fully react at room temperature for 2-2.5h, then dialyze with PBS solution containing 5mM EDTA for 6-8H, Remove unreacted free 2-IT (dialysis membrane 1000MCW).

[0052] (3) The same amount of thiolated Rotuximab was mixed with 11B8, and the MPEGS-PEI solution (mAb / PEI=1000:3.44) prepared in (1) was...

Embodiment 2

[0053] Example 2: SDS-PAGE Identification of Anti-CD20 Antibody Nanoclusters

[0054] The prepared anti-CD20 antibody nanoclusters were subjected to SDS-PAGE electrophoresis with 8% separating gel, and stained with Coomassie brilliant blue to identify the molecular weight of the anti-CD20 antibody nanoclusters.

Embodiment 3

[0055] Example 3: Particle size determination and morphology observation of anti-CD20 antibody nanoclusters

[0056] After the prepared anti-CD20 antibody nanoclusters were diluted with sterilized PBS, the particle size was detected by a dynamic tube scattering instrument (ALV / CGS-3, Germany).

[0057] The diluted sample was placed on a special copper grid for transmission electron microscopy, and after air-drying, it was negatively stained with 2% PTA (phosphotungstic acid) solution, and its morphology was observed with a transmission electron microscope.

[0058] The diluted sample can also be placed under an atomic force microscope for morphological observation by conventional methods.

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Abstract

The present invention relates to the preparation of a new generation of multifunctional antibody nanoclusters and their synergistic therapeutic applications. The antibody nanoclusters are composed of two or more antibodies of the same type or different types coupled to the same macromolecular chain through a hinge Or on the sphere, the hinge agent molecules linking the antibody and the macromolecular chain include chemical bonds that can be broken by redox degradation, hydrolysis, acidolysis, etc., and chemical bonds that cannot be broken. Its advantages are as follows: for the first time, two or different antibodies were cross-linked in vitro and successfully applied in vivo, which solved the scientific problem that antibody cross-linking with synergistic therapeutic function could not be applied in vivo. The CD20 antibody nanocluster successfully activates four pathways of anti-tumor effects of all currently known CD20 antibodies in vitro experiments. The antibody nanocluster has stronger anti-tumor effect, longer half-life and stronger killing effect.

Description

technical field [0001] The invention relates to the technical field of medicines, in particular to the preparation of a new generation of multifunctional antibody nanoclusters and their synergistic therapeutic applications. Background technique [0002] Molecular targeted therapy refers to treatments aimed at cell signal transduction and other biological pathways involved in tumorigenesis and development. The targets of molecular targeted therapy in a broad sense include any submolecules from DNA to protein levels involved in multiple processes such as cell differentiation, apoptosis, migration, invasive behavior, lymphatic metastasis, and systemic metastasis. Since this century, molecular targeted therapy has become the focus and main direction of oncology research, promoting the development of tumor treatment concepts and theories. Rituximab (Rituximab, C2B8, trade name MabThera) is the first FDA-approved monoclonal antibody (monoclonal antibody, mAb) targeting human CD20...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/44A61K47/68A61P35/00A61K47/60
Inventor 李威孙赟赵赫陈迪张歌张莉祝仙弟
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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