An antibody-coupled drug targeting on EGFR, a preparation method thereof, and uses thereof

An antibody-conjugated drug, antibody technology, applied in the direction of antibodies, anti-tumor drugs, drug combinations, etc., to achieve the effects of good repeatability, considerable yield, stable and controllable quality

Active Publication Date: 2019-01-15
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, antibody therapy has certain limitations, and it is usu

Method used

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  • An antibody-coupled drug targeting on EGFR, a preparation method thereof, and uses thereof
  • An antibody-coupled drug targeting on EGFR, a preparation method thereof, and uses thereof
  • An antibody-coupled drug targeting on EGFR, a preparation method thereof, and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1V

[0051] The synthesis of embodiment 1VC-MMAE (MC-VC-PAB-MMAE):

[0052] 1.1 Synthesis of MMAE

[0053] Synthetic route such as figure 1 as shown in a:

[0054] Step a: Synthesis of Boc-Dap-N-desmethylephedrine

[0055] Compound Boc-Dap-OH (500mg, 1.7mmol) and (1S, 2R)-(±)-desmethylephedrine (289.3mg, 1.9mmol) were dissolved in 10mL DMF, followed by adding DEPC (368.9mg, 2.2 mmol ) and Et 3 N (228.8mg.2.2mmol), the reaction was stirred overnight at room temperature. After the reaction was completed, add water to stop the reaction, extract three times with ethyl acetate, and extract once with saturated NaCl solution, anhydrous NaCl 2 SO 4 Let dry overnight. Separation and purification by column chromatography, the separation condition was petroleum ether: ethyl acetate = 5:1, and 485 mg of white solid was obtained. C 23 h 36 N 2 o 5 .MS(ESI)m / z:321. 1 H NMR (400MHz, DMSO-d 6 )δ7.66(d,J=8.6Hz,1H),7.36–7.25(m,4H),7.22–7.16(m,1H), 5.37(d,J=4.6Hz,1H),4.46(t,J =5.1Hz,1H...

Embodiment 2

[0074] Example 2 Preparation of Antibody Drug Conjugate LR004-VC-MMAE

[0075] Include the following steps:

[0076] 1) The MC-VC-PAB-MMAE prepared in Example 1 (that is, the linker-cytotoxic drug) was dissolved in dimethyl sulfoxide to obtain a linker-cytotoxic drug stock solution.

[0077] 2) TCEP reducing agent is dissolved in buffer solution PBS (containing 1mM DTPA), and a certain molar amount of reducing agent stock solution is prepared.

[0078] 3) Replace the buffer of LR004 antibody before reduction, and the buffer system is PBS (containing 1mM DTPA). The replacement method is to replace the desalting column. The replacement method is as follows: load 2.5 mL on the desalting column, elute 3.5 mL with replacement buffer, concentrate the concentration by ultrafiltration, ultrafiltration speed 4000 rpm, and centrifuge at 4 ° C for 10 min. Configure to obtain 5mg / mL LR004 antibody solution.

[0079] 4) Mix the 5 mg / mL LR004 antibody solution in step 3) with the reduci...

Embodiment 3

[0085] Example 3 Detection of EGFR expression in different tumor cells

[0086] 3.1 Western Blot detection of EGFR expression levels in different tumor cells

[0087] Collect logarithmic phase esophageal cancer cells (KYSE450), breast cancer cells (MDA-MB-468), head and neck cancer cells (SCC-25), colon cancer cells (HCT-116), pancreatic cancer cells (AsPC-1), Osteosarcoma cells (143B) and prostate cancer cells (PC-3). Cells were digested, collected and counted, collecting 1×10 7 The above cells were centrifuged and the supernatant was discarded for later use. Pre-cool RIPA protein extraction reagent, add protease inhibitor (phosphorylated protein needs to add phosphatase inhibitor at the same time). Add 0.1M PMSF stock solution before the start of protein extraction, and the final concentration of PMSF is 1mM. For cell counting, the number of cells is 1 x 10 7 Add 1ml of lysate, centrifuge at 4°C, store, and measure protein concentration by BCA method. The Western Blot ...

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Abstract

The invention discloses an antibody coupling drug targeting on EGFR, a preparation method thereof and uses thereof. The antibody-conjugated drug targeting EGFR is named LR004-VC-MMAE consisting of anantibody, a cytotoxic drug and a linker, wherein the antibody drug conjugate has a structure represented by the formula I, wherein mAb is an LR004 monoclonal antibody, n=2-8. The novel antibody-conjugated drug LR004-VC-MMAE can not only target EGFR antigen, but also has strong cytotoxicity to tumor cells. Compared with LR004 itself, it did not affect the affinity, endocytosis and targeting of theantibody, and better retained its biological function. Compared to LR004, the antitumor effect of LR004-VC-MMAE antibody-conjugated drug is significantly improved, and the tumor disappeared. Comparedto LR004, LR004-VC-MMAE antibody-conjugated total antibody showed longer half-life, slower clearance rate, lower concentration of free MMAE in plasma, shorter half-life and faster clearance rate, which is conducive to reduce toxicity.

Description

technical field [0001] The invention belongs to the field of biotechnology medicine. Specifically, the present invention provides a class of antibody-conjugated drugs that can produce targeted tumor killing effects, their preparation methods and uses. technical background [0002] According to the "Global Cancer Report", in 2018, it is estimated that there will be 18.1 million new cancer cases and 9.6 million deaths in the world, with an average of 18 cancer deaths every minute, and the global cancer burden will further increase. China is a big cancer country, and the number of new cases is on the rise. Among them, solid tumors such as lung cancer, breast cancer, and colorectal cancer are cancers with a very high incidence rate, and usually when diagnosed, the disease has already developed to the middle and late stages. At present, the main treatment methods are surgical treatment, radiotherapy and drug chemotherapy. This treatment strategy not only has a poor prognosis an...

Claims

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Application Information

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IPC IPC(8): A61K47/68A61K47/65A61K47/55A61K38/07A61K39/395A61P35/00
CPCA61K38/07A61K39/39558A61K47/55A61K47/65A61K47/6813A61K47/6849A61P35/00
Inventor 李卓荣胡馨月苗庆芳王蓉金洁刘秀均崔阿龙李毅甄永苏
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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