Antibody-coupled drug as well as preparation and application thereof

An antibody-conjugated drug and antibody technology, applied in the field of medicine, can solve the problems affecting the pharmacokinetic evaluation and clinical application of ADC drugs, the inability to obtain drug-loading sites to determine ADC products, and the difficulty of ADCs to effectively reach tumor cells, etc. Achieve the effect of clear structure of drug loading site, increase effective drug delivery rate, and increase tumor cell penetration rate

Active Publication Date: 2015-07-08
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the ADC studies adopt the non-selective coupling mode with uncontrollable drug loading (Sammet, B., Steinkuhler, C., Sewald, N. Antibody-drug conjugates in tumor therapy. Pharmaceutical Patent Analyst. 2012, 1:65 -73.), leading to the inability to obtain ADC products with definite drug-loading sites and drug-loading ratios, which seriously affects the pharmacokinetic evaluation and clinical application of ADC drugs
In addition, the molecular weight of the commonly used complete antibody is relatively large, usually above 100kDa. The excessive molecular volume makes it difficult for the ADC to effectively reach the tumor cells, resulting in a low effective drug delivery rate.

Method used

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  • Antibody-coupled drug as well as preparation and application thereof
  • Antibody-coupled drug as well as preparation and application thereof
  • Antibody-coupled drug as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] In this implementation, the F(ab') obtained by pepsin degradation 2 The fragment is reduced with reducing agent β-mercaptoethylamine (β-MEA), the specific method and steps are as follows:

[0069] (1) Degradation of anti-CD20 antibody with pepsin

[0070] Weigh 5 mg of anti-CD20 antibody and dissolve it in 2 mL of sodium acetate buffer solution, and measure the concentration value. Add pepsin solution, the mass ratio of anti-CD20 antibody to pepsin is 20:1, react at 37°C for 12h, adjust the pH value to 8.0, stop the reaction, and separate by SDS-PAGE (sodium dodecyl sulfate polyacrylamide Gel electrophoresis) to detect the progress of the reaction and the purity of the sample, such as figure 1 As shown, the results showed that the anti-CD20 antibody was degraded by pepsin to obtain F(ab') 2 fragment.

[0071] (2) Using a reducing agent to convert F(ab') 2 Fragment restore

[0072] F(ab') 2 Fragment concentration is 1.63mg / mL, β-MEA is used as reducing agent, when t...

Embodiment 2

[0078] In this implementation, the F(ab') obtained by pepsin degradation 2 The fragment is reduced with reducing agent β-mercaptoethylamine (β-MEA), the specific method and steps are as follows:

[0079] (1) Degradation of anti-CD20 antibody with pepsin

[0080] Weigh 5 mg of anti-CD20 antibody and dissolve it in 2 mL of sodium acetate buffer solution, and measure the concentration value. Add pepsin solution, the mass ratio of anti-CD20 antibody to pepsin is 20:1, react at 37°C for 12h, adjust the pH value to 8.0, and terminate the reaction to obtain F(ab’) 2 fragment.

[0081] (2) Using a reducing agent to convert F(ab') 2 Fragment restore

[0082] F(ab') 2 Fragment concentration is 0.5mg / mL, β-MEA is used as reducing agent, when the concentration is 10mM, react at room temperature, start timing when adding reducing agent, respectively at 5, 10, 20, 30, 60, Sampling at 90 minutes, removing the reducing agent through a desalting column, and then using SDS-PAGE to detect ...

Embodiment 3

[0086] In this embodiment, the Fab'-PEG-DOX (doxorubicin) conjugated drug is prepared by the following method, which specifically includes the following steps:

[0087] (1) Degradation of anti-CD20 antibody with pepsin

[0088] Weigh 5 mg of anti-CD20 antibody and dissolve it in 2 mL of sodium acetate buffer solution, and measure the concentration value. Add pepsin solution, the mass ratio of anti-CD20 antibody to pepsin is 20:1, react at 37°C for 12h, adjust the pH value to 8.0, terminate the reaction, and obtain F(ab’) 2 fragment.

[0089] (2) Using a reducing agent to convert F(ab') 2 Fragment restore

[0090] Take the F(ab') obtained in step (1) 2 To 2 mL of the solution, 1.54 mg of solid reducing agent β-MEA was added (the concentration of β-MEA was 10 mM), and the reaction was allowed to stand at room temperature for 30 min. After the reaction is completed, the mixture is separated and purified to obtain a Fab' fragment with reduced disulfide bonds in the hinge regi...

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Abstract

The invention discloses an antibody-coupled drug as well as a preparation and the application thereof. The antibody-coupled drug is obtained from a heresy-base bifunctional-group polyethylene glycol covalent coupled drug molecule and a Fab' fragment, wherein the structure of the antibody-coupled drug is shown in formula I: Fab'-heresy base bifunctional group polyethylene glycol-drug molecule (1), wherein the Fab' is a fragment of an anti-CD20 antibody and the drug molecule is a cytotoxic drug molecule. According to the antibody-coupled drug, the F(ab')2 fragment degraded from the anti-CD20 antibody is reduced to a Fab' fragment of which the hinge area comprises two free thiols; and the Fab' fragment is then covalently coupled with the drug molecule by the heresy-base bifunctional-group polyethylene glycol, so that the antibody-coupled drug is obtained. The antibody-coupled drug prepared by the invention has the characteristics of fixed drug loading ratio and clear drug loading site structure; the antibody-coupled drug also retains the binding capacity of the anti-CD20 antibody to CD20 proteins, so that the antibody-coupled drug has targeting ability to the CD20-positive cells and is applicable to treatment of non-Hodgkin lymphomas.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to an antibody-coupled drug and its preparation method and application. Background technique [0002] Tumor is an important disease that threatens human health. The current treatment methods mainly include surgery, radiotherapy, chemotherapy and other means. The high recurrence rate, tumor metastasis and corresponding complications after surgical treatment are the main reasons for the failure of surgical treatment. For radiotherapy and chemotherapy, while killing tumor cells, it will inevitably cause damage to normal human tissue cells, resulting in serious side effects. Therefore, the indiscriminate killing effect of radiotherapy / chemotherapy also severely limits their clinical application. application. [0003] Broad-spectrum anti-tumor drugs such as doxorubicin used in tumor treatment can interfere with or block the proliferation of cells by inhibiting the synthesis of cellular ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/48A61P35/00
Inventor 张竞周展苏志国马光辉
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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