Antibody prodrug and preparation method and application thereof

A technology of prodrugs and antibodies, applied in the direction of antibodies, anti-tumor drugs, drug combinations, etc., can solve the problems of lack of targeting

Pending Publication Date: 2022-02-25
TAIZHOU MABTECH PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] However, the Probody drug lacks targeting before it enters the inflammatory site of the tumor tissue. Only when the drug comes into cont

Method used

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  • Antibody prodrug and preparation method and application thereof
  • Antibody prodrug and preparation method and application thereof
  • Antibody prodrug and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of targeted antibody prodrugs

[0054] Use genetic engineering technology to synthesize part A with targeted protein gene sequence, such as VEGF single-chain antibody gene sequence, the light chain variable region has the nucleotide sequence shown in SEQ ID NO:1 and the amino acid shown in SEQ ID NO:2 Sequence, the heavy chain variable region has the nucleotide sequence shown in SEQ ID NO:3 and the amino acid sequence shown in SEQ ID NO:4.

[0055] Using genetic engineering technology to synthesize part C of the antibody gene sequence with therapeutic effect, such as the complete antibody gene sequence of EGFR, its light chain has the nucleotide sequence shown in SEQ ID NO:5 and the amino acid sequence shown in SEQ ID NO:6, Its heavy chain has the nucleotide sequence shown in SEQ ID NO:7 and the amino acid sequence shown in SEQ ID NO:8.

[0056] The connecting peptide part B connecting part A and part C has the amino acid sequence shown in ASLSG...

Embodiment 2

[0059] Example 2: Cell culture, expression and purification of targeted antibody prodrugs

[0060] The CHO host cells transfected with the targeting antibody prodrug were cultured, expressed and purified without serum to obtain the targeting antibody prodrug CMAB0301.

[0061] Part A of the targeted antibody prodrug CMAB0301 is a VEGF single-chain antibody whose light chain variable region has the amino acid sequence shown in SEQ ID NO: 2, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 4; part B is a connecting peptide containing a urokinase-type plasminogen activation substrate, which has the amino acid sequence shown in ASLSGRSDNHGSAS; part C has the amino acid sequence shown in SEQ ID NO: 6 for the light chain, and the amino acid sequence shown in SEQ ID NO: 8 for the heavy chain. Whole antibody to EGFR with amino acid sequence shown.

[0062] The targeted antibody prodrug CMAB0301 adopts basic culture and supplementary culture methods ...

Embodiment 3

[0070] Example 3: In vitro activity detection of targeted antibody prodrugs

[0071] The targeting antibody prodrug CMAB0301 was obtained by the above method, and the binding and affinity detection of VEGF and EGFR were performed.

[0072] (1) VEGF binding assay (ELISA):

[0073] Coat the recombinant VEGF165 antigen (1 μg / ml) on a 96-well ELISA plate, add 100 μl to each well overnight at 4°C; block the ELISA plate with PBS containing 10% BSA at 37°C for 1 h; Add CMAB0301 serially diluted from high concentration, incubate at 37°C for 1 h, wash with PBS containing 0.1% Tween-20 three times; add horseradish peroxidase-coupled goat anti-human F(ab')2 The secondary antibody (1:1,000) was incubated for 30 min; TMB chromogenic solution was added, the color was developed for 10 min, the reaction was terminated with 1M sulfuric acid, and the absorbance value (OD value) was measured at 450 nm with a microplate reader.

[0074] The binding ability of targeting antibody prodrug CMAB0301...

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Abstract

The invention provides a targeting antibody prodrug which is composed of a part A, a part B and a part C. The part A is a protein with targeting performance, the part C is an antibody/antibody receptor fusion protein with a therapeutic effect, the part A is connected with the part C through the part B, the part B is connected with the part C through the part C, and the part A with the targeting protein is connected to the antigen binding region of the part C antibody.

Description

technical field [0001] The invention belongs to the field of biotechnology, and discloses an antibody prodrug, its preparation method and application, more specifically, the invention discloses a targeted antibody prodrug, its preparation method and application. Background technique [0002] Tumor refers to the new organism formed by local tissue hyperplasia under the action of various tumorigenic factors. Tumor cells will undergo metabolic changes different from normal cells. Transformation to adapt to changes in the metabolic environment. In a similar situation, some autoimmune diseases or other diseases also have some local or systemic inflammatory / metabolic abnormal reactions. [0003] In the treatment of tumors, in addition to conventional surgery, radiotherapy, chemotherapy, biological therapy, and traditional Chinese medicine treatment, different targeted therapy techniques can be used for targeted therapy according to the different targets of tumors at the organ tis...

Claims

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Application Information

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IPC IPC(8): C07K16/46A61K39/44A61P35/00A61P29/00
CPCC07K16/22C07K16/2863A61K39/44A61P35/00A61P29/00C07K2317/31C07K2319/50
Inventor 王皓陶静张大鹏
Owner TAIZHOU MABTECH PHARM CO LTD
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