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Preparation method of antibody coupled drug

A technology of antibody-drug coupling and monoclonal antibody, which is applied in the field of biomedicine, can solve the problem of high toxicity and achieve the effect of increasing the growth inhibition rate

Inactive Publication Date: 2019-03-22
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
  • Preparation method of antibody coupled drug
  • Preparation method of antibody coupled drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Preparation and optimization of antibody conjugate drug HK02-MMAE

[0046] 1. Optimization of reduction conditions

[0047] 1. Take agarose gel G25FF column, first equilibrate with 5 column volumes of pH7.4, 0.01M PBS buffer, then load the anti-human ErbB2 monoclonal antibody (ie naked antibody), and then use 1 The column volume was eluted with a pH7.4, 0.01M PBS buffer containing 2mM EDTA to obtain a purified antibody solution.

[0048] 2. After completing step 1, take the purified antibody solution and check the purity of the purified antibody by SDS-PAGE.

[0049] The results show that the purified antibody solution is electrophoretic pure.

[0050] 3. After completing step 1, take the purified antibody solution and use the BCA method to determine the concentration.

[0051] 4. Mix an aqueous solution containing 10-50mmol / L TCEP and 1mM EDTA with 1mL purified antibody solution (concentration of 5mg / mL) to obtain a mixed solution. In the mixed solution, the molar ra...

Embodiment 2

[0079] Example 2. Scale-up production of antibody conjugate drug HK02-MMAE

[0080] The scale-up production (approximately 200 mg) of the antibody conjugate drug HK02-MMAE was carried out according to the following production conditions:

[0081] Reduction conditions: the concentration of purified antibody is 5mg / mL, the molar ratio of purified antibody and TCEP is 1:3, room temperature;

[0082] Coupling conditions: The molar ratio of vc-MMAE and the reduced purified antibody is 6:1, the concentration of reduced purified antibody is 2.5mg / mL, the reduction time is 60min, and the coupling time is 1h, which will complete the reduction step. The mixed solution was diluted 2 times with pH7.4, 0.01M PBS buffer.

[0083] Detect the purity of antibody conjugated drug HK02-MMAE and the average number of drug conjugates per antibody molecule.

[0084] See the results of testing the purity of the antibody conjugated drug HK02-MMAE Figure 4 Middle A and Table 2. The results showed that the pur...

Embodiment 3

[0090] Example 3. ELISA to detect the affinity of antibody-conjugated drug HK02-MMAE to antigen (human ErbB2 protein)

[0091] T6-17 cells (described in the following documents: Wang Xian, Xie Xiaoqiang, Cao Liang, etc.. Expression of tumor protein P185 in the supernatant of T6-17 cell culture. Journal of Cellular and Molecular Immunology, 2008, 24(10): 1018 -1019) is a stable transgenic cell line that transfers human ErbB2 gene into NIH3T3 cells (mouse embryo fibroblasts), and its cell membrane surface highly expresses human ErbB2 protein.

[0092] Cell lysate: pH 7.5, 50 mM Tris-HCl buffer containing 150 mM NaCl, 1% (v / v) Triton-100 and 4 mM protease inhibitor Complete.

[0093] Washing solution (pH7.4) (ie PBST buffer): add KH to distilled water 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O2.9g, NaCl 8g, KCl0.2g and Tween-20 0.05% (volume percentage), dilute to 1L.

[0094] The antibody to be tested is the antibody-conjugated drug HK02-MMAE or anti-human ErbB2 monoclonal antibody.

[0095] 1. Di...

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Abstract

The invention discloses a preparation method of an antibody coupled drug. The preparation method comprises the following steps: mixing an anti-human ErbB2 monoclonal antibody and a reducer for a reduction reaction; diluting a reduced reaction system with a 0.005-0.015 M PBS buffer solution, the pH of which is 7.2-7.6, for 2-4 times to obtain a diluted solution; and mixing the diluted solution withvc-MMAE for a coupling reaction to obtain the antibody coupled drug. Experiments verify that the antibody coupled drug and T-DM1 can inhibit proliferation of SKBR3, NCI-N87, HCC1954, MDA-MB453 and KPL-4 and are constant in inhibiting effect substantially. The effect of the antibody coupled drug in inhibiting JIMT-1 proliferation is obviously higher than that of T-DM1. The preparation method has important application value.

Description

Technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a method for preparing antibody-conjugated drugs. Background technique [0002] Human ErbB2 protein (ErbB2 for short, also known as HER2 or P185) is a glycoprotein that belongs 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. 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). Tumors with high ErbB2 expression often have poor prognosis. This shows that ErbB2 has become one of the important targets of tumor targeted therapy. [0003] Targeted therapy of tumors is an important means to treat tumors. A successful strategy is to use ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/68A61K38/07A61P35/00
CPCA61K38/07A61K47/6817A61K47/6855A61K47/6863A61P35/00
Inventor 程联胜赵群刘兢张中伟
Owner HEFEI HANKEMAB BIOTECH CO LTD
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