A pro-antibody conjugated drug targeting tumor cells expressing EGFR and its application

An antibody-conjugated drug, tumor cell technology, used in anti-tumor drugs, drug combinations, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin and other directions, can solve serious side effects and other problems, achieve low toxicity Side effects, high potency effects

Active Publication Date: 2020-01-21
TAIZHOU MABTECH PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibody-drug conjugates targeting EGFR are potential therapeutic agents, however, there are still risks associated with serious side effects

Method used

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  • A pro-antibody conjugated drug targeting tumor cells expressing EGFR and its application
  • A pro-antibody conjugated drug targeting tumor cells expressing EGFR and its application
  • A pro-antibody conjugated drug targeting tumor cells expressing EGFR and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, preparation of proantibody conjugated drug

[0045]According to the patent US 2011 / 0003969, PanP-DM1 was prepared by coupling DM1-SMCC with the lysine residue of antibody PanP. The main process is as follows: 5mg / mL antibody was mixed with 6.4mg / mL DM1-SMCC, the reaction buffer was phosphate buffer (50mM, PH=7.5) containing 2mM EDTA and 50mM Nacl, and the reaction time was 2 hours. According to the above method, Pan was also coupled with DM1-SMCC to form Pan-DM1.

[0046] In addition, the anti-TNFα antibody was coupled with DM1 to prepare a control antibody-conjugated drug. According to the patent US2010 / 0092495, after measuring the absorbance of the antibody-conjugated drug at 252nm and 280nm by ultraviolet spectrophotometry, the coupling ratio of the drug-antibody is calculated. The distribution of drugs was determined by reversed-phase ultra-high performance liquid chromatography-tandem mass spectrometry (RP-UPLC / ESI-MS). Under reducing and non-reduc...

experiment example 1

[0053] Experimental example 1, PDC activity detection

[0054] EGFR binding determined by ELISA and flow cytometry

[0055] EGFR-Fc was coated onto a 96-well plate and blocked with PBS containing 10% skimmed milk powder. After washing, add the indicated concentration of antibody and incubate at room temperature for 1 hour. After washing, add HRP-labeled anti-human F(ab')2, incubate at 37°C for 30min, and finally add TMB substrate solution. Absorbance was read at 450 nm and fitted using a 4 parameter curve.

[0056] When performing flow cytometry analysis, A431 cells overexpressing EGFR were mixed with the antibody to be tested in FACS buffer (PBS buffer containing 1% fetal bovine serum) and incubated for 1 hour. After washing, FITC-labeled anti-human secondary antibody was added and incubated for 30 minutes. Next, wash again with FACS buffer and analyze on BD's FACSCalibur system.

[0057] PanP-DM1 showed similar enzyme activation properties to PanP.

[0058] To further ...

experiment example 2

[0060] Experimental example 2, PDC cytotoxicity test in vitro

[0061] The effect of PanP and PanP-DM1 conjugates on tumor cell viability was evaluated using CCK-8 kit. Spread the cells cultured with 1% calf serum into a 96-well cell culture plate (5000 per well for BT-474, MCF-7 and DiFi cells; 3000 per well for H292 cells; 2000 per well for A431 cells ), cultured overnight to allow it to adhere to the wall. The next day, corresponding concentrations of activated PanP, activated PanP-DM1 or anti-TNFα-DM1 antibody as a control group were added to each well and incubated for different times (48 hours for A431 cells, 72 hours for other cells). Cell viability was determined using CCK-8 kit.

[0062] Calculate the survival cell ratio according to the following formula: [(A450 value of experimental group-A450 value of background group) / (A450 value of untreated group-A450 value of background group)]*100

[0063] Comparing the inhibitory activity of PanP-DM1, activated PanP-DM1 an...

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Abstract

The present invention discloses a probody conjugating drug for targeting tumor cells expressing EGFR, and applications thereof. According to the present invention, a maytansine derivative (DM1) is conjugated to PanP through a stable non-reducing thioether bond to obtain the probody conjugating drug PanP-DM1 for targeting tumor cells expressing EGFR; and the probody conjugating drug PDC has high potency and high tumor selectivity, and the experiment results prove that the probody conjugating drug has low toxic-side effects on normal tissues.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention discloses a proantibody-conjugated drug targeting tumor cells expressing EGFR and an application thereof. Background technique [0002] Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase that plays an important role in tumor progression. Excessive EGFR activation is associated with tumorigenesis and metastasis. It is reported that 60%-80% of colon cancers have overexpression of EGFR. In addition, EGFR is also an important marker for predicting colorectal cancer and breast cancer. More importantly, EGFR is located on the surface of cancer cells, so it is an ideal target for the development of targeted antibodies. Antibodies can specifically bind to the extracellular domain III domain of EGFR, which not only prevents ligand binding, but also prevents dimerization of extended domains on domain II (Li S, et al. Structural basis for ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/68A61K31/537A61P35/00C07K16/28
Inventor 钱卫珠
Owner TAIZHOU MABTECH PHARM CO LTD
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