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Conjugate of anti-human DLL4 humanized antibody and maytansine alkaloid DM1, preparation method and application thereof

A technology of humanized antibodies and alkaloids, which is applied in the direction of medical preparations with non-active ingredients, medical preparations containing active ingredients, drug combinations, etc., can solve inhomogeneity, product heterogeneity, difficult to control drug and Issues such as antibody coupling position and stoichiometry, to achieve the effects of growth inhibition, high affinity activity, and promotion of tumor targeting

Active Publication Date: 2020-08-07
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Traditional ADCs drugs couple cytotoxin molecules to the surface of antibodies through the natural lysine (Lys) residues on the antibody surface or the sulfhydryl groups of cysteine ​​(Cys) generated by reducing interchain disulfide bonds, but these two Both methods are difficult to control the coupling position and stoichiometry of the drug and antibody. Therefore, the ADCs prepared by random coupling have serious product heterogeneity, and the drug / antibody ratios (drug / antibody ratios, DAR) uneven

Method used

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  • Conjugate of anti-human DLL4 humanized antibody and maytansine alkaloid DM1, preparation method and application thereof
  • Conjugate of anti-human DLL4 humanized antibody and maytansine alkaloid DM1, preparation method and application thereof
  • Conjugate of anti-human DLL4 humanized antibody and maytansine alkaloid DM1, preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0060]The engineered anti-human DLL4 humanized antibody THL4 obtained by site-directed mutation of the anti-human DLL4 humanized antibody H3L2 was prepared by molecular biology techniques: (1) determined according to the nucleic acid sequences of the light chain and heavy chain of the humanized antibody H3L2 Corresponding antibody light chain 207 (valine) and heavy chain 121 (lysine), design primers (a total of 8), mutate to cysteine ​​corresponding nucleotides by Overlap-PCR; (2 ) for T-A cloning, transforming the target fragment into the E. coli DH5α host strain, picking a single clone of the strain for DNA sequencing, and sequencing the correct sequence to pMH3 (neomycin resistance) and pCA-puro (neomycin resistance) and pCA-puro ( Puromycin resistance) two expression plasmids, after DNA sequencing was correct, four engineering plasmids were obtained, named pCA-tH, pMH3-tH, pCA-tL and pMH3-tL; (3) Transient transfection of plasmids into 293F Fermentation in cells, the ferme...

Embodiment 2

[0063] The coupling between DM1 and the engineered anti-human DLL4 humanized antibody THL4 was detected by high performance liquid chromatography (HPLC).

[0064] Agilent 1200HPLC was used to analyze the conjugation of antibody-drug conjugate HLmD4. 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.

[0065] The experimental results see figure 2 , compared with H3L2 whose main peak appeared at 9.min, HLmD4 also appeared at 11min, 14min, 16.5min and 19min respectively, corresponding to the number of small molecules coupled to DM1 were 2, 4, 6 and 8, and the corresponding pea...

Embodiment 3

[0067] Affinity detection of antibody-drug conjugate HLmD4 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 The conjugates HLmD4 and H3L2 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.

[0068] The experimental results see image 3 , compared with H3L2, the affinity of HLmD4 to DLL4 antigen is slightly lower,...

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Abstract

The invention relates to the technical field of biopharmacy, discloses a conjugate of an anti-human DLL4 (Delta like 4) humanized antibody and maytansine alkaloid DM1, a preparation method and application thereof. A coupled conjugate of the novel anti-human DLL4 humanized antibody THL4 and the maytansine alkaloid DM1 is obtained. The preparation method and the application are characterized in thatthe small molecular toxin maytansine alkaloid DM1 is coupled to an engineered anti-human DLL4 humanized antibody THL4 in a fixed-point manner to synthesize the conjugate. The conjugate HLmD4 is usedfor promoting the tumor targeting of toxin molecule DM1, reducing the toxic action on normal cells of a body and achieving a better treatment effect.

Description

technical field [0001] The present invention is in the field of antibody conjugated drugs. Specifically, the present invention relates to site-directed mutation on the anti-human DLL4 humanized antibody H3L2 to obtain an engineered antibody, which is site-directedly coupled to a small toxin molecule maytansine alkaloid DM1 conjugate. Background technique [0002] DLL4 (Delta like 4) is an important ligand of Notch receptor in the evolutionarily conserved Notch signaling pathway. DLL4 ligand participates in the regulation of proliferation, apoptosis and differentiation of adjacent cells by binding to Notch receptors on the surface of adjacent cells And biological processes such as stem cell proliferation and renewal. Not only that, DLL4 is overexpressed in human tumor tissues and participates in the development of tumor blood vessels. So far, researchers have observed the overexpression of DLL4 in tumor vessels such as kidney cancer, bladder cancer, colon cancer, brain tumo...

Claims

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Application Information

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IPC IPC(8): A61K47/68A61K47/54A61K31/5365A61P35/00A61P35/02
CPCA61K47/6843A61K47/545A61K31/5365A61P35/00A61P35/02
Inventor 吴旻王旻王世静费文仪赵玉红温慧冯宇琪匡璐
Owner CHINA PHARM UNIV
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