Anti-human DLL4 monoclonal antibody and aplysiatoxin derivative MMAE conjugate

A monoclonal antibody, dolastatin technology, applied in the direction of drug combination, anti-tumor drugs, medical preparations with non-active ingredients, etc., can solve the problem of high toxicity, achieve the effect of low toxicity, ensure controllability, and improve curative effect

Inactive Publication Date: 2017-11-24
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

MMAE is not used alone as a drug for cancer treatment due to its high toxicity and non-selectivity

Method used

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  • Anti-human DLL4 monoclonal antibody and aplysiatoxin derivative MMAE conjugate
  • Anti-human DLL4 monoclonal antibody and aplysiatoxin derivative MMAE conjugate
  • Anti-human DLL4 monoclonal antibody and aplysiatoxin derivative MMAE conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]Preparation of antibody-drug conjugate MvM03 by coupling anti-human DLL4 monoclonal antibody MMGZ01 with dolastatin derivative MMAE: (1) Antibody part: MMGZ01 was purified by Protein A column affinity chromatography, and the above-mentioned The purified MMGZ01 antibody was replaced by agarose gel G25 FF column molecular sieve chromatography; the antibody concentration was determined by BCA method; 4-12% SDS-PAGE gel electrophoresis verified that the purified product was electrophoretic pure; the reducing agent TCEP was mixed with The above-mentioned antibodies were thoroughly mixed at a molar ratio of 3:1, and after reacting at 4°C for 1 hour, the above-mentioned mixture was desalted by agarose gel G25 FF column molecular sieve chromatography with a pH 7.0 PBS solution containing 1M DTPA; (2) coupling Product part: Dissolve vc-MMAE powder in DMSO solution fully, mix vc-MMAE solution with the above antibody product at a molar ratio of 15:1, stir gently, and react on ice fo...

Embodiment 2

[0035] The coupling between MMAE and anti-human DLL4 monoclonal antibody MMGZ01 was detected by high performance liquid chromatography (HPLC).

[0036] Agilent 1200 HPLC was used to analyze the conjugation of antibody-drug conjugate MvM03. Sample detection conditions are as follows: (1) mobile phase A: 20mmol / L PBS (pH 7.0)+1.5mol / L ammonium sulfate; (2) mobile phase B: 20mmol / L PBS (pH7.0) / isopropanol=7.5 / 2.5; (3) Elution gradient: 10-100% B; (4) Elution time: 20min; (5) Flow rate: 0.60mL / min; (6) Injection volume: 10uL; (7) Detection wavelength: 280nm. According to the number of peaks and the area of ​​each peak, the antibody-drug conjugation ratio (DAR) was calculated proportionally.

[0037] See the experimental results figure 1 , compared with MMGZ01 whose main peak appeared at 9.5min, MvM03 also appeared at 12.5min, 15min, 17min and 18min respectively, corresponding to the number of coupled MMAE small molecules were 2, 4, 6 and 8 , and the corresponding peak area ra...

Embodiment 3

[0039] Affinity detection of antibody-drug conjugate MvM03 and human DLL4 antigen: by ELISA method, 1 μg / mL DLL4 antigen was added to 96-well ELISA plate at 100 μL per well, and coated overnight at 4°C; after washing the plate three times with PBS, the Conjugates MvM03 and MMGZ01 of Example 1 were added to the 96-well plate with concentration gradients of 0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, and 1000 nM according to the blank group, and incubated at 37°C for 2 hours ; After washing the plate three times with PBS, add goat anti-mouse IgG antibody coupled with horseradish peroxidase (HRP), and incubate at 37°C for 1 hour; after washing the plate three times with PBS, add TMB chromogenic solution, React at room temperature in the dark for 20 minutes, and finally add stop solution to terminate the reaction, and detect OD450-OD630 with a microplate reader.

[0040] See the experimental results figure 2 , compared with MMGZ01, the affinity of MvM03 to DLL4 antigen is slig...

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Abstract

The invention discloses an anti-human DLL4 monoclonal antibody and aplysiatoxin derivative MMAE conjugate, which relates to the technical field of biological pharmacy. The invention is characterized by comprising a preparation method of the conjugate of micromolecule toxin aplysiatoxin derivative MMAE and the anti-human DLL4 monoclonal antibody MMG201 as well as a purpose thereof. The method employs anti-human DLL4 monoclonal antibody MMG201 and selects a vcMMAE joint and a pharmaceutical composition, a phosphine reducing agent tricarboxyethyl phosphine TCEP is used for partially reducing the antibody MMG201 during a coupling technology, then the antibody MMG201 is subjected to coupling with vcMMAE and is subjected to conjugate purifying, through optimization of the process, a novel anti-human DLL4 monoclonal antibody medical conjugate MvM03 is prepared, the novel anti-human DLL4 monoclonal antibody medical conjugate MvM03 has the beneficial effect that the tumour targeting of toxin molecule MMAE is promoted, the poisoning effect on body normal cell is reduced, and the good treatment effect can be achieved.

Description

technical field [0001] The present invention is in the field of antibody conjugated drugs. Specifically, the present invention relates to a conjugate of anti-human DLL4 monoclonal antibody MMGZ01 coupled with a small toxin molecule dolastatin derivative MMAE. Background technique [0002] DLL4 (Delta like 4) is an important ligand of Notch receptor in the Notch signaling pathway. DLL4 participates in the regulation of proliferation, apoptosis, differentiation of adjacent cells, proliferation and renewal of stem cells by binding to Notch receptors on the surface of adjacent cells. biological process. In addition, DLL4 is overexpressed in human tumor tissues and participates in the development of tumor blood vessels. So far, overexpression of DLL4 has been observed in tumor blood vessels such as kidney cancer, bladder cancer, colon cancer, brain tumor and breast cancer. . In recent years, a number of studies have found that the DLL4 / Notch signaling pathway plays an importan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/68A61K47/65A61K38/07A61P35/00
CPCA61K38/07
Inventor 王旻吴旻王世静
Owner CHINA PHARM UNIV
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