Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Anti-CD20 monoclonal antibody-aplysiatoxin conjugate as well as preparation method and application thereof

A monoclonal antibody and Aplysia toxin technology, applied in the field of biotechnology and medicine, can solve the problems of tumor cell lethality to be improved, cumbersome operation steps, etc., achieve enhanced anti-tumor effect, reduce toxic and side effects, and improve anti-tumor activity Effect

Active Publication Date: 2014-08-13
ZHEJIANG UNIV
View PDF3 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Since this preparation method requires site-directed mutation of the OFA monoclonal antibody, the operation steps are cumbersome, and the anti-CD20 antibody obtained after mutation can only be coupled with 1 to 2 dolastatin molecules, and the lethality to tumor cells needs to be improved

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Anti-CD20 monoclonal antibody-aplysiatoxin conjugate as well as preparation method and application thereof
  • Anti-CD20 monoclonal antibody-aplysiatoxin conjugate as well as preparation method and application thereof
  • Anti-CD20 monoclonal antibody-aplysiatoxin conjugate as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 Preparation of OFA-vcMMAE conjugates

[0046] The preparation method of OFA-vcMMAE conjugate comprises:

[0047] (1) Add 5.5-10 times the molar amount of tris(β-chloroethyl)phosphate (TCEP) to 10mg / mL OFA monoclonal antibody (dissolved in PBS, pH7.4), bathe in water at 37°C for 2h, and place on ice superior;

[0048] (2) While stirring, add 10 times excess vcMMAE dissolved in 30% acetonitrile / water (synthesized by Jiangyin Kangnuotai Biotechnology Co., Ltd., 10 times excess means that the molar amount of vcMMAE in the reaction system is greater than or equal to 10 times that of OFA monoclonal antibody. times), react at 4°C for 60 min, and add excess cysteine ​​to terminate the reaction;

[0049] (3) Pass through a HiPrepTM26 / 10 desalting column, elute with PBS, remove small molecules, and obtain the conjugate OFA-vcMMAE; the obtained conjugate is sterilized by a water membrane with a pore size of 0.22 μm, and stored at -20°C for later use.

[0050] The c...

Embodiment 2

[0053] The biological activity detection of embodiment 2 conjugates

[0054] Next, CD20 positive cells Raji, Daudi and WIL2-S and CD20 negative cell K562 were used as objects to detect the biological activity of OFA and OFA-vcMMAE conjugates.

[0055] 1 Flow affinity detection

[0056] (1) Take 1×10 6 WIL2-S cells were incubated with different concentrations (10, 3.33, 1.11, 0.37, 0.12 μg / mL) of OFA monoclonal antibody or OFA-vcMMAE in 1% BSA (dissolved in PBS) solution at 4°C for 30 min;

[0057] (2) After washing twice with PBS, add FITC-labeled goat anti-human IgG (H+L) polyclonal antibody (diluted 1:200) and incubate at 4°C for 30 min; after washing with PBS, measure the average fluorescence intensity of the cells by flow cytometry (MFI).

[0058] The combination of OFA or OFA-vcMMAE and CD20-positive cells WIL2-S was detected by flow cytometry, and the strength of the binding force was shown by the average fluorescence intensity of FITC after secondary antibody labelin...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an anti-CD20 monoclonal antibody-aplysiatoxin conjugate as well as a preparation method and application thereof. The preparation method comprises the steps of dissolving the anti-CD20 monoclonal antibody into a buffer solution, adding trichloroethyl phosphate to carry out reduction reaction; mixing restored anti-CD20 monoclonal antibody with aplysiatoxin having a connecting arm to carry out coupled reaction; and separating and purifying to obtain the anti-CD20 monoclonal antibody-aplysiatoxin conjugate after reaction is finished. Compared with the prior art, 8 aplysiatoxin molecules are coupled in the conjugate disclosed by the invention at most; the anti-CD20 monoclonal antibody-aplysiatoxin conjugate has the biological functions of the anti-CD20 monoclonal antibody and MMAE, has strong tumor targeting property, can kill CD20 positive tumor cells, and also reduces the toxic and side effects caused by independent dosing of the MMAE; and the anti-tumor effect is obviously reinforced.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and in particular relates to an anti-CD20 monoclonal antibody-dolastatin conjugate and a preparation method and application thereof. Background technique [0002] CD20 is a unique membrane surface protein on the surface of B lymphocytes and B lymphoma cells, which is not expressed in hematopoietic stem cells, primitive B cells, normal plasma cells and other normal tissues (Bubien J K et al., J Cell Biol, 1993, 121 (5): 1121-1132). Therefore, the development of monoclonal antibodies against CD20 antigen can specifically kill B cells and B lymphoma cells and achieve good therapeutic effects. [0003] Ofatumumab (OFA for short) is a fully humanized targeting anti-CD20 monoclonal antibody, which was approved by the FDA in 2009 for refractory chronic lymphocytic leukemia (CLL) ineffective in the treatment of fludarabine and alemtuzumab (CASTILLO J .Expert Opin Investing Drμgs, 2009, 18(4):...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K1/107A61K38/07A61K47/48A61P35/00
Inventor 陈枢青李朝辉张骞徐晓倩
Owner ZHEJIANG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com