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Conjugate of anti-human DLL4 (Delta-Like Ligand 4) monoclonal antibody and adriamycin

A monoclonal antibody and conjugate technology, applied in the direction of anti-tumor drugs, drug combinations, medical preparations containing active ingredients, etc., to achieve the effects of good solubility, controllability, and mild reaction conditions

Inactive Publication Date: 2017-10-27
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no relevant patents and literature reports on anti-human DLL4 antibody-drug conjugates in China. Therefore, the inventor hopes to couple the anti-human DLL4 monoclonal antibody MMGZ01 with a toxin small molecule drug, and make full use of the targeting properties of the antibody. The toxicity of small toxin molecules improves the efficacy and reduces side effects

Method used

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  • Conjugate of anti-human DLL4 (Delta-Like Ligand 4) monoclonal antibody and adriamycin
  • Conjugate of anti-human DLL4 (Delta-Like Ligand 4) monoclonal antibody and adriamycin
  • Conjugate of anti-human DLL4 (Delta-Like Ligand 4) monoclonal antibody and adriamycin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Preparation of antibody-drug conjugate MvM03 conjugated by anti-human DLL4 monoclonal antibody MMGZ01 and doxorubicin DOX: (1) Antibody part: MMGZ01 was purified by ProteinA column affinity chromatography, and the above-mentioned purified MMGZ01 was purified by PBS with pH 7.0 The antibody was replaced with the antibody solution system by agarose gel G25FF column molecular sieve chromatography; the antibody concentration was determined by the BCA method; 4-12% SDS-PAGE gel electrophoresis was used to verify that the purified product was electrophoretic pure; the reducing agent TCEP was mixed with the above antibody at 3 The molar ratio of : 1 was fully mixed, and after reacting for 1 hour at 4°C, the above mixture was desalted by agarose gel G25FF column molecular sieve chromatography with a pH 7.0 PBS solution containing 1M DTPA; (2) toxin part: take toxin DOX powder Fully dissolve in DMSO solution, take 2.8 mg of GMBS powder and dissolve in 1 mL of DMSO solution, mix t...

Embodiment 2

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

[0036] Agilent 1200HPLC was used to analyze the conjugation of antibody-drug conjugate MGD03. 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=7.0 / 3.0; (3) Elution gradient: 10-100% B; (4) Elution time: 25min; (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 11min, MGD03 also appeared at 14.5min, 17.5min, 20min and 21.5min respectively, corresponding to the number of coupled DOX small molecules were 2, 4, 6 and 8, and the corresponding peak area pro...

Embodiment 3

[0039] Affinity detection between antibody drug conjugate MGD03 and human DLL4 antigen: Add 1 μg / mL DLL4 antigen at 100 μL per well to a 96-well microtiter plate by ELISA method, and coat at 4°C overnight; after washing the plate three times with PBS, put The conjugates MvM03 and MMGZ01 of Example 1 were added to the 96-well plate in the blank group with concentration gradients of 0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, and 1000 nM, 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 MGD03 to DLL4 antigen is slightl...

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Abstract

The invention discloses an antibody drug conjugate MGD03 of an anti-human DLL4 (Delta-Like Ligand 4) monoclonal antibody MMGZ01 and adriamycin DOX and relates to the technical field of bio-pharmaceuticals. The invention is characterized by further comprising a preparation method and application of a conjugate of micro-molecule toxin adriamycin DOX and the anti-human DLL4 monoclonal antibody MMGZ01. The preparation method disclosed by the invention comprises the following steps: adopting the anti-human DLL4 monoclonal antibody MMGZ01, selecting the joint and pharmaceutical composition of GMBS (Gamma-Maleimido-butyryloxy Succinimide Ester)-DOX, reducing the antibody MMGZ01 in the conjugating process by using a tricarboxyethyl phosphine (TCEP) part of a phosphine reducing agent, conjugating with the GMBS-DOX, and purifying the conjugate, thereby obtaining the novel drug conjugate MGD03 of the anti-human DLL4 monoclonal antibody MMGZ01 by optimizing the process. The conjugate MGD03 disclosed by the invention has the beneficial effects that the tumor targeting property of the toxin molecule DOX is promoted, the toxic actions on normal cells of the organism are reduced, and an excellent treatment effect is 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 doxorubicin DOX. 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 important role in t...

Claims

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Application Information

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IPC IPC(8): A61K47/68A61K31/704A61P35/00
Inventor 王旻吴旻王世静
Owner CHINA PHARM UNIV
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