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Preparation method of antibody coupled drug

An antibody-drug conjugated and conjugated technology, applied in the field of biomedicine, can solve the problem of high toxicity and achieve the effect of improving the growth inhibition rate

Inactive Publication Date: 2019-04-30
HEFEI HANKEMAB BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because MMAE is highly toxic and non-selective, it cannot be used as a drug alone for cancer treatment

Method used

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  • Preparation method of antibody coupled drug
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  • Preparation method of antibody coupled drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Preparation of anti-human ErbB2 bispecific antibody As3H

[0047] The sequence of the anti-human ErbB2 monoclonal antibody Trastuzumab (IgG1, κ) comes from the patent US5821337 of Genentech, and the anti-human ErbB2 monoclonal antibody HuA21 was created by Hefei Hankemaibo Biotechnology Co., Ltd. Its sequence comes from the patent ZL201410489895X.

[0048] As3H is a ScFv formed by connecting the variable regions of the light and heavy chains of HuA21 to the N-terminus of the heavy chain of Herceptin through a linker with a sequence of GGGGSGGGGSGGGGS. The amino acid sequence of the variable regions of the light and heavy chains in the ScFv is GGGGSGGGGSGGGGSGGGGS, and the anti-human ErbB2 double The schematic diagram of the structure of the specific antibody As3H is as follows figure 1 shown.

[0049] The construction process of the recombinant vector used to express the anti-human ErbB2 bispecific antibody As3H is as follows:

[0050] According to reports ...

Embodiment 2

[0054] Example 2. Preparation and condition optimization of antibody-conjugated drug As3H-MMAE

[0055] 1. Take a Sephadex G25FF column, equilibrate with 5 column volumes of pH7.4, 0.01M PBS buffer, then load the anti-human ErbB2 bispecific antibody As3H (ie, naked antibody), and then use Purified antibody solution was obtained by eluting with 1 column volume of 2 mM EDTA in pH 7.4, 0.01 M PBS buffer.

[0056] 2. After completing step 1, take the purified antibody solution and check the purity of the purified antibody by SDS-PAGE.

[0057] The results showed that the purified antibody solution was electrophoresis pure.

[0058] 3. After completing step 1, take the purified antibody solution and measure the concentration by BCA method.

[0059] 4. Mix an aqueous solution containing 10-50 mmol / L TCEP and 1 mM EDTA with 1 mL of purified antibody solution (concentration of 5 mg / mL) to obtain a mixed solution. In the mixture, the molar ratio of purified antibody and TCEP is 1:4,...

Embodiment 3

[0071]Embodiment 3, the scale-up production of antibody-conjugated drug As3H-MMAE

[0072] The scale-up production (about 200 mg) of antibody-conjugated drug As3H-MMAE is carried out according to the following production conditions:

[0073] Reduction conditions: the concentration of purified antibody is 5mg / mL, the molar ratio of purified antibody to TCEP is 1:5, room temperature;

[0074] Coupling conditions: the molar ratio of vc-MMAE and the reduced purified antibody is 7:1, the concentration of the reduced purified antibody is 2.5mg / mL, the reduction time is 60min, and the coupling time is 30min, the reduction step will be completed The mixture was diluted 2 times with pH7.4, 0.01M PBS buffer.

[0075] The purity of the antibody-conjugated drug As3H-MMAE and the average drug-conjugated number per antibody molecule were detected.

[0076] For the results of detecting the purity of the antibody-conjugated drug As3H-MMAE, see image 3 Middle A and Table 2. The results sh...

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Abstract

The invention discloses a preparation method of an antibody coupled drug. The preparation method comprises following steps: mixing an anti-human ErbB2 bi-specific antibody with a reducing agent to carry out reduction reactions; after reduction, using a PBS buffer solution (pH=7, 0.01M) to dilute the reaction system by 2 to 4 times, and mixing the diluent with vc-MMAE to carry out coupling reactions to obtain the antibody coupled drug. The amino acid sequence of the anti-human ErbB2 bi-specific antibody is represented by SEQ ID No.1; and the amino acid sequence of the light chain is representedby SEQ ID No.2. The experiment results show that the antibody coupled drug and T-DM1 can inhibit the proliferation of SKBR3, NCI-N87, HCC1954, MDA-MB-453, and KPL-4, the inhibition effects are basically the same, and the JIMT-1 inhibiting effect of the antibody coupled drug is better than that of T-DM1. The preparation method has an important application value.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a preparation method of an antibody conjugated drug. Background technique [0002] Human ErbB2 protein (abbreviated ErbB2, also known as HER2 or P185) is a glycoprotein belonging to the epidermal growth factor receptor (ErbB / HER) family in the type I receptor tyrosine kinase family. ErbB2 is closely related to the occurrence and development of tumors, and high expression of ErbB2 proto-oncogene or ErbB2 can be detected in a variety of cancers (such as breast cancer, lung cancer, pancreatic cancer, and ovarian cancer). At the same time, ErbB2 is often used as an important prognostic indicator for some tumors (such as breast cancer), and tumors with high ErbB2 expression often have poor prognosis. It can be seen that ErbB2 has become one of the important targets of tumor targeted therapy. [0003] Targeted therapy of tumors is an important means of treating tumors. A succe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61P35/00A61K38/07
CPCA61K38/07A61K39/39558A61K39/39566A61P35/00A61K2300/00
Inventor 程联胜赵群刘兢张中伟
Owner HEFEI HANKEMAB BIOTECH CO LTD
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